Herbicide-tolerant protox genes produced by DNA shuffling

ABSTRACT

The present invention provides novel DNA sequences coding for protoporphyrinogen oxidase (protox) enzymes from soybean, wheat, cotton, sugar beet, oilseed rape, rice, sorghum, and sugar cane. In addition, the present invention teaches modified forms of protox enzymes that are herbicide tolerant . Plants expressing herbicide tolerant protox enzymes taught herein are also provided. These plants may be engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or they may be transformed with a gene encoding an herbicide tolerant form of a plant protox enzyme. The present invention further provides shuffled DNA molecules encoding protox enzymes having enhanced tolerance to a herbicide that inhibits the protox activity encoded by a template DNA molecule from which the shuffled DNA molecule is derived.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/059,164, filed Apr. 13, 1998, which is acontinuation-in-part of U.S. application Ser. No. 09/050,603, filed Mar.30, 1998, which is a continuation-in-part of U.S. application Ser. No.08/808,931, filed Feb. 28, 1997, which is a continuation-in-part of U.S.application Ser. No. 08/472,028, filed Jun. 6, 1995, now U.S. Pat. No.5,767,373, issued Jun. 16, 1998, which is a continuation-in-part of U.S.application Ser. No. 08/261,198, filed Jun. 16, 1994, now abandoned.Said U.S. application Ser. No. 08/808,931 also claims the benefit ofU.S. Provisional Application No. 60/012,705, filed Feb. 28, 1996, U.S.Provisional Application No. 60/013,612, filed Feb. 28, 1996, and U.S.Provisional Application No. 60/020,003, filed Jun. 21, 1996. Said U.S.application Ser. No. 09/059,164 is also a continuation-in-part of U.S.application Ser. No. 09/038,878, filed Mar. 11, 1998. All of theaforementioned applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to DNA molecules encodingherbicide-tolerant forms of the enzyme protoporphyrinogen oxidase(“protox”). The invention further relates to herbicide-tolerant plantsas well as methods for tissue culture selection and herbicideapplication based on these herbicide-tolerant forms of protox.

BACKGROUND OF THE INVENTION

[0003] I. The Protox Enzyme and its Involvement in the Chlorophyll/HemeBiosynthetic Pathway

[0004] The biosynthetic pathways that lead to the production ofchlorophyll and heme share a number of common steps. Chlorophyll is alight harvesting pigment present in all green photosynthetic organisms.Heme is a cofactor of hemoglobin, cytochromes, P450 mixed-functionoxygenases, peroxidases, and catalyses (see, e.g. Lehninger,Biochemistry, Worth Publishers, New York (1975)), and is therefore anecessary component for all aerobic organisms.

[0005] The last common step in chlorophyll and heme biosynthesis is theoxidation of protoporphyrinogen IX to protoporphyrin IX.Protoporphyrinogen oxidase (referred to herein as “protox”) is theenzyme that catalyzes this last oxidation step (Matringe et al.,Biochem. J. 260: 231 (1989)).

[0006] The protox enzyme has been purified either partially orcompletely from a number of organisms including the yeast Saccharomycescerevisiae (Labbe-Bois and Labbe, In Biosynthesis of Heme andChlorophyll, E. H. Dailey, ed. McGraw Hill: New York, pp. 235-285(1990)), barley etioplasts (Jacobs and Jacobs, Biochem. J. 244: 219(1987)), and mouse liver (Dailey and Karr, Biochem. 26: 2697 (1987)).Genes encoding protox have been isolated from two prokaryotic organisms,Escherichia coli (Sasarman et al., Can. J. Microbiol. 39: 1155 (1993))and Bacillus subtilis (Dailey et al., J. Biol. Chem. 269: 813 (1994)).These genes share no sequence similarity; neither do their predictedprotein products share any amino acid sequence identity. The E. coliprotein is approximately 21 kDa, and associates with the cell membrane.The B. subtilis protein is 51 kDa, and is a soluble, cytoplasmicactivity.

[0007] Protox encoding genes have now also been isolated from humans(see Nishimura et al., J. Biol. Chem. 270(14): 8076-8080 (1995) andplants (International application no. PCT/IB95/00452 filed Jun. 8, 1995,published Dec. 21, 1995 as WO 95/34659).

[0008] II. The Protox Gene as a Herbicide Target

[0009] The use of herbicides to control undesirable vegetation such asweeds or plants in crops has become an almost universal practice. Therelevant market exceeds a billion dollars annually. Despite thisextensive use, weed control remains a significant and costly problem forfarmers.

[0010] Effective use of herbicides requires sound management. Forinstance, time and method of application and stage of weed plantdevelopment are critical to getting good weed control with herbicides.Since various weed species are resistant to herbicides, the productionof effective herbicides becomes increasingly important. Novel herbicidescan now be discovered using high-throughput screens that implementrecombinant DNA technology. Metabolic enzymes essential to plant growthand development can be recombinantly produced though standard molecularbiological techniques and utilized as herbicide targets in screens fornovel inhibitors of the enzymes' activity. The novel inhibitorsdiscovered through such screens may then be used as herbicides tocontrol undesirable vegetation.

[0011] Unfortunately, herbicides that exhibit greater potency, broaderweed spectrum and more rapid degradation in soil can also have greatercrop phytotoxicity. One solution applied to this problem has been todevelop crops that are resistant or tolerant to herbicides. Crop hybridsor varieties resistant to the herbicides allow for the use of theherbicides without attendant risk of damage to the crop. Development ofresistance can allow application of a herbicide to a crop where its usewas previously precluded or limited (e.g. to pre-emergence use) due tosensitivity of the crop to the herbicide. For example, U.S. Pat. No.4,761,373, incorporated herein by reference, is directed to plantsresistant to various imidazolinone or sulfonamide herbicides. Theresistance is conferred by an altered acetohydroxyacid synthase (AHAS)enzyme. U.S. Pat. No. 4,975,374, incorporated herein by reference,relates to plant cells and plants containing a gene encoding a mutantglutamine synthetase (GS) resistant to inhibition by herbicides thatwere known to inhibit GS, e.g. phosphinothricin and methioninesulfoximine. U.S. Pat. No. 5,013,659, incorporated herein by reference,is directed to plants that express a mutant acetolactate synthase (ALS)that renders the plants resistant to inhibition by sulfonylureaherbicides. U.S. Pat. No. 5,162,602, incorporated herein by reference,discloses plants tolerant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The tolerance is conferred byan altered acetyl coenzyme A carboxylase(ACCase). U.S. Pat. No.5,554,798, incorporated herein by reference, discloses transgenicglyphosate resistant maize plants, which tolerance is conferred by analtered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene.

[0012] The protox enzyme serves as the target for a variety ofherbicidal compounds. The herbicides that inhibit protox include manydifferent structural classes of molecules (Duke et al., Weed Sci. 39:465 (1991); Nandihalli et al., Pesticide Biochem. Physiol. 43: 193(1992); Matringe et al., FEBS Lett. 245: 35 (1989); Yanase and Andoh,Pesticide Biochem. Physiol. 35: 70 (1989)). These herbicidal compoundsinclude the diphenylethers {e.g. acifluorfen,5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid; its methylester; or oxyfluorfen,2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles,(e.g. oxidiazon,3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one),cyclic imides (e.g.S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and itsO-phenylpyrrolidino- and piperidinocarbamate analogs. Many of thesecompounds competitively inhibit the normal reaction catalyzed by theenzyme, apparently acting as substrate analogs.

[0013] Typically, the inhibitory effect on protox is determined bymeasuring fluorescence at about 622 to 635 nm, after excitation at about395 to 410 nM (see, e.g. Jacobs and Jacobs, Enzyme 28: 206 (1982);Sherman et al., Plant Physiol. 97:280 (1991)). This assay is based onthe fact that protoporphyrin IX is a fluorescent pigment, andprotoporphyrinogen IX is nonfluorescent.

[0014] The predicted mode of action of protox-inhibiting herbicidesinvolves the accumulation of protoporphyrinogen IX in the chloroplast.This accumulation is thought to lead to leakage of protoporphyrinogen IXinto the cytosol where it is oxidized by a peroxidase activity toprotoporphyrin IX. When exposed to light, protoporphyrin IX can causeformation of singlet oxygen in the cytosol. This singlet oxygen can inturn lead to the formation of other reactive oxygen species, which cancause lipid peroxidation and membrane disruption leading to rapid celldeath (Lee et al., Plant Physiol. 102: 881 (1993)).

[0015] Not all protox enzymes are sensitive to herbicides that inhibitplant protox enzymes. Both of the protox enzymes encoded by genesisolated from Escherichia coli (Sasarman et al., Can. J. Microbiol. 39:1155 (1993)) and Bacillus subtilis (Dailey et al., J. Biol. Chem. 269:813 (1994)) are resistant to these herbicidal inhibitors. In addition,mutants of the unicellular alga Chlamydomonas reinhardtii resistant tothe phenylimide herbicide S-23142 have been reported (Kataoka et al., J.Pesticide Sci. 15: 449 (1990); Shibata et al., In Research inPhotosynthesis, Vol. III, N. Murata, ed. Kluwer:Netherlands. pp. 567-570(1992)). At least one of these mutants appears to have an altered protoxactivity that is resistant not only to the herbicidal inhibitor on whichthe mutant was selected, but also to other classes of protox inhibitors(Oshio et al., Z. Naturforsch. 48c: 339 (1993); Sato et al., in ACSSymposium on Porphyric Pesticides, S. Duke, ed. ACS Press: Washington,D.C. (1994)). A mutant tobacco cell line has also been reported that isresistant to the inhibitor S-21432 (Che et al.,. Z. Naturforsch. 48c:350 (1993).

[0016] III. Plastid Transformation and Expression

[0017] Plastid transformation, in which genes are inserted by homologousrecombination into some or all of the several thousand copies of thecircular plastid genome present in each plant cell, takes advantage ofthe enormous copy number advantage over nuclear-expressed genes topermit expression levels that may exceed 10% of the total soluble plantprotein. In addition, plastid transformation is desirable because inmost plants plastid-encoded traits are not pollen transmissible; hence,potential risks of inadvertent transgene escape to wild relatives oftransgenic plants is obviated. Plastid transformation technology isextensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818,and 5,576,198; in PCT Application Nos. WO 95/16783 and WO 97/32977; andin McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7305 (1994), allof which are incorporated herein by reference. Plastid transformationvia biolistics was achieved initially in the unicellular green algaChlamydomonas reinhardtii (Boynton et al. (1988) Science 240: 1534-1537,incorporated herein by reference) and this approach, using selection forcis-acting antibiotic resistance loci (spectinomycin/streptomycinresistance) or complementation of non-photosynthetic mutant phenotypes,was soon extended to Nicotiana tabacum (Svab et al. (1990) Proc. Natl.Acad. Sci. USA 87: 8526-8530, incorporated herein by reference).

[0018] The basic technique for tobacco chloroplast transformationinvolves the particle bombardment of leaf tissue or PEG-mediated uptakeof plasmid DNA in protoplasts with regions of cloned plastid DNAflanking a selectable antibiotic resistance marker. The 1 to 1.5 kbflanking regions, termed “targeting sequences,” facilitate homologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the 156 kb tobacco plastid genome.Initially, point mutations in the chloroplast 16S rDNA and rps12 genesconferring resistance to spectinomycin and/or streptomycin were utilizedas selectable markers for transformation (Svab, Z., Hajdukiewicz, P.,and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub,J. M., and Maliga, P. (1992) Plant Cell 4, 39-45, incorporated herein byreference). This resulted in stable homoplasmic transformants at afrequency of approximately one per 100 bombardments of target leaves.The presence of cloning sites between these markers allowed creation ofa plastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, P., EMBO J. 12: 601-606 (1993), incorporated herein byreference). Substantial increases in transformation frequency wereobtained by replacement of the recessive rRNA or r-protein antibioticresistance genes with a dominant selectable marker, the bacterial aadagene encoding the spectinomycin-detoxifying enzymeaminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P. (1993)Proc. Natl. Acad. Sci. USA 90, 913-917, incorporated herein byreference). Previously, this marker had been used successfully forhigh-frequency transformation of the plastid genome of the green algaChlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. AcidsRes. 19, 4083-4089, incorporated herein by reference). Recently, plastidtransformation of protoplasts from tobacco and the moss Physcomitrellapatens has been attained using polyethylene glycol (PEG) mediated DNAuptake (O'Neill et al. (1993) Plant J. 3: 729-738; Koop et al. (1996)Planta 199: 193-201, both of which are incorporated herein byreference).

SUMMARY OF THE INVENTION

[0019] The present invention provides DNA molecules isolated from wheat,soybean, cotton, sugar beet, oilseed rape, rice, sorghum, and sugar caneencoding enzymes having protoporphyrinogen oxidase (protox) activity andchimeric genes comprising such DNA. Sequences of such DNA molecules areset forth in SEQ ID NOs:9 (wheat), 11 (soybean), 15 (cotton), 17 (sugarbeet), 19 (oilseed rape), 21 (rice), 23 (sorghum), and 36 (sugar cane).

[0020] The present invention also provides modified forms of plantprotoporphyrinogen oxidase (protox) enzymes that are resistant tocompounds that inhibit unmodified naturally occurring plant protoxenzymes, and DNA molecules coding for such inhibitor-resistant plantprotox enzymes. Thus, in one aspect the present invention provides a DNAmolecule encoding a plant protox enzyme that is capable of beingincorporated into a DNA construct used to transform a plant containingwild-type, herbicide-sensitive protox, wherein the DNA molecule has atleast one point mutation relative to a wild-type DNA molecule encodingplant protox such that upon transformation with the DNA construct theplant contains the DNA molecule, which renders the plant resistant tothe application of a herbicide that inhibits naturally occurring plantprotox. The present invention includes chimeric genes and modified formsof naturally occurring protox genes that can express theinhibitor-resistant plant protox enzymes in plants.

[0021] Genes encoding inhibitor-resistant plant protox enzymes can beused to confer resistance to protox-inhibitory herbicides in wholeplants and as a selectable marker in plant cell transformation methods.Accordingly, the present invention also includes plants, including thedescendants thereof, plant tissues and plant seeds containing plantexpressible genes encoding these modified protox enzymes. These plants,plant tissues and plant seeds are resistant to protox-inhibitors atlevels that normally are inhibitory to the naturally occurring protoxactivity in the plant. Plants encompassed by the invention especiallyinclude those that would be potential targets for protox inhibitingherbicides, particularly agronomically important crops such as maize andother cereal crops such as barley, wheat, sorghum, rye, oats, turf andforage grasses, millet and rice. Also comprised are other crop plantssuch as sugar cane, soybean, cotton, sugar beet, oilseed rape andtobacco.

[0022] The present invention accordingly provides a method for selectingplant cells transformed with a DNA molecule of the invention thatencodes a herbicide-tolerant form of plant protox. The method comprisesintroducing the DNA molecule into plant cells whose growth is sensitiveto inhibition by herbicides to which the protox encoded by the DNAmolecule is resistant, thus forming a transformed plant cell. Thetransformed plant cell whose growth is resistant to the selectedherbicide is identified by selection at a herbicide concentration thatinhibits the growth of untransformed plant cells.

[0023] The present invention is directed further to methods for theproduction of plants, including plant material, such as for exampleplant tissues, protoplasts, cells, calli, organs, plant seeds, embryos,pollen, egg cells, zygotes, together with any other propagating materialand plant parts, such as for example flowers, stems, fruits, leaves,roots originating in transgenic plants or their progeny previouslytransformed by means of the process of the invention, which produce aninhibitor-resistant form of the plant protox enzyme provided herein.Such plants may be stably transformed with a structural gene encodingthe resistant protox, or prepared by direct selection techniques wherebyherbicide resistant lines are isolated, characterized and developed.

[0024] In another aspect, the present invention is directed to a methodfor controlling unwanted vegetation growing at a locus where aherbicide-tolerant, agronomically useful plant, which is transformedwith a DNA molecule according to the present invention that encodes aherbicide-tolerant form of plant protox, has been cultivated. The methodcomprises applying to the locus to be protected an effective amount ofherbicide that inhibits naturally occurring protox activity.

[0025] The present invention is further directed to probes and methodsfor detecting the presence of genes encoding inhibitor-resistant formsof the plant protox enzyme and quantitating levels ofinhibitor-resistant protox transcripts in plant tissue. These methodsmay be used to identify or screen for plants or plant tissue containingand/or expressing a gene encoding an inhibitor-resistant form of theplant protox enzyme.

[0026] The present invention also relates to plastid transformation andto the expression of DNA molecules in a plant plastid. In a preferredembodiment, a native plant protox enzyme or a modified plant protoxenzyme is expressed in plant plastids to obtain herbicide resistantplants.

[0027] In a further embodiment, the present invention is directed to achimeric gene comprising: (a) a DNA molecule isolated from a plant,which in its native state encodes a polypeptide that comprises a plastidtransit peptide, and a mature enzyme that is natively targeted to aplastid of the plant by the plastid transit peptide, wherein the DNAmolecule is modified such that it does not encode a functional plastidtransit peptide; and (b) a promoter capable of expressing the DNAmolecule in a plastid, wherein the promoter is operatively linked to theDNA molecule. The DNA molecule may be modified in that at least aportion of the native plastid transit peptide coding sequence is absentfrom the DNA molecule. Alternatively, the DNA molecule may be modifiedin that one or more nucleotides of the native plastid transit peptidecoding sequence are mutated, thereby rendering an encoded plastidtransit peptide nonfunctional. The present invention also relates toplants homoplasmic for chloroplast genomes containing such chimericgenes. In a preferred embodiment, the DNA molecule encodes an enzymethat is naturally inhibited by a herbicidal compound. In this case, suchplants are resistant to a herbicide that naturally inhibits the enzymeencoded by a DNA molecule according to the present invention.

[0028] The present invention is also directed to plants made resistantto a herbicide by transforming their plastid genome with a DNA moleculeaccording to the present invention and to methods for obtaining suchplants. In a preferred embodiment, the DNA molecule encodes an enzymethat is naturally inhibited by a herbicidal compound. In a morepreferred embodiment, the DNA molecule encodes an enzyme havingprotoporphyrinogen oxidase (protox) activity, which is modified so thatit that confers resistance to protox inhibitors. A further embodiment ofthe present invention is directed to a method for controlling the growthof undesired vegetation, which comprises applying to a population of theabove-described plants an effective amount of an inhibitor of theenzyme.

[0029] The present invention also provides a novel method for selectinga transplastomic plant cell, comprising the steps of: introducing theabove-described chimeric gene into the plastome of a plant cell;expressing the encoded enzyme in the plastids of the plant cell; andselecting a cell that is resistant to a herbicidal compound thatnaturally inhibits the activity of the enzyme, whereby the resistantcell comprises transformed plastids. In a preferred embodiment, theenzyme is naturally inhibited by a herbicidal compound and thetransgenic plant is able to grow on an amount of the herbicidal compoundthat naturally inhibits the activity of the enzyme. In a furtherpreferred embodiment, the enzyme has protoporphyrinogen oxidase (protox)activity and is modified so that it that confers resistance to protoxinhibitors.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0030] SEQ ID NO:1: DNA coding sequence for an Arabidopsis thalianaprotox-1 protein.

[0031] SEQ ID NO:2: Arabidopsis protox-1 amino acid sequence encoded bySEQ ID NO: 1.

[0032] SEQ ID NO:3: DNA coding sequence for an Arabidopsis thalianaprotox-2 protein.

[0033] SEQ ID NO:4: Arabidopsis protox-2 amino acid sequence encoded bySEQ ID NO:3.

[0034] SEQ ID NO:5: DNA coding sequence for a maize protox-1 protein.

[0035] SEQ ID NO:6: Maize protox-1 amino acid sequence encoded by SEQ IDNO:5.

[0036] SEQ ID NO:7: DNA coding sequence for a maize protox-2 protein.

[0037] SEQ ID NO:8: Maize protox-2 amino acid sequence encoded by SEQ IDNO:7.

[0038] SEQ ID NO:9: Partial DNA coding sequence for a wheat protox-1protein.

[0039] SEQ ID NO:10: Partial wheat protox-1 amino acid sequence encodedby SEQ ID NO:9.

[0040] SEQ ID NO:11: DNA coding sequence for a soybean protox-1 protein.

[0041] SEQ ID NO:12: Soybean protox-1 protein encoded by SEQ ID NO:11.

[0042] SEQ ID NO:13: Promoter sequence from Arabidopsis thalianaprotox-1 gene.

[0043] SEQ ID NO:14: Promoter sequence from maize protox-1 gene.

[0044] SEQ ID NO:15: DNA coding sequence for a cotton protox-1 protein.

[0045] SEQ ID NO:16: Cotton protox-1 amino acid sequence encoded by SEQID NO:15.

[0046] SEQ ID NO:17: DNA coding sequence for a sugar beet protox-1protein.

[0047] SEQ ID NO:18: Sugar beet protox-1 amino acid sequence encoded bySEQ ID NO: 17.

[0048] SEQ ID NO:19: DNA coding sequence for an oilseed rape protox-1protein.

[0049] SEQ ID NO:20: Oilseed rape protox-1 amino acid sequence encodedby SEQ ID NO: 19.

[0050] SEQ ID NO:21: Partial DNA coding sequence for a rice protox-1protein.

[0051] SEQ ID NO:22: Partial rice protox-1 amino acid sequence encodedby SEQ ID NO:21.

[0052] SEQ ID NO:23: Partial DNA coding sequence for a sorghum protox-1protein.

[0053] SEQ ID NO:24: Partial sorghum protox-1 amino acid sequenceencoded by SEQ ID NO:23.

[0054] SEQ ID NO:25: Maize protox-1 intron sequence.

[0055] SEQ ID NO:26: Promoter sequence from sugar beet protox-1 gene.

[0056] SEQ ID NO:27: Pclp_P1a—plastid clpP gene promoter top strand PCRprimer.

[0057] SEQ ID NO:28: Pclp_P1b—plastid clpP gene promoter bottom strandPCR primer.

[0058] SEQ ID NO:29: Pclp_P2b—plastid clpP gene promoter bottom strandPCR primer.

[0059] SEQ ID NO:30: Trps16_P1a—plastid rps16 gene top strand PCRprimer.

[0060] SEQ ID NO:31: Trps16_p1b—plastid rps16 gene bottom strand PCRprimer.

[0061] SEQ ID NO:32: minpsb_U—plastid psbA gene top strand primer.

[0062] SEQ ID NO:33: minpsb_L—plastid psbA gene bottom strand primer.

[0063] SEQ ID NO:34: APRTXP1a—top strand PCR primer.

[0064] SEQ ID NO:35: APRTXP1b—bottom strand PCR primer.

[0065] SEQ ID NO:36: Partial DNA coding sequence for a sugar caneprotox-1 protein.

[0066] SEQ ID NO:37: Partial sugar cane protox-1 amino acid sequenceencoded by SEQ ID NO:36.

DEPOSITS

[0067] The following vector molecules have been deposited withAgricultural Research Service, Patent Culture Collection (NRRL),Northern Regional Research Center, 1815 North University Street, Peoria,Ill. 61604, U.S.A on the dates indicated below:

[0068] Wheat protox-1a, in the pBluescript SK vector, was deposited Mar.19, 1996, as pWDC-13 (NRRL#B21545).

[0069] Soybean protox-1, in the pBluescript SK vector, was depositedDec. 15, 1995 as pWDC-12 (NRRL#B-21516).

[0070] Cotton protox-1, in the pBluescript SK vector, was deposited Jul.1, 1996 as pWDC-15 (NRRL #B-21594).

[0071] Sugar beet protox-1, in the pBluescript SK vector, was depositedJul. 29, 1996, as pWDC-16 (NRRL #B-21595N).

[0072] Oilseed rape protox-1, in the pBluescript SK vector, wasdeposited Aug. 23, 1996, as pWDC-17 (NRRL #B-21615).

[0073] Rice protox-1, in the pBluescript SK vector, was deposited Dec.6, 1996, as pWDC-18 (NRRL#B-21648).

[0074] Sorghum protox-1, in the pBluescript SK vector, was depositedDec. 6, 1996, as pWDC-19 (NRRL #B-21649).

[0075]

[0076] Resistant mutant pAraC-2Cys, in the pMut-1 plasmid, was depositedon Nov. 14, 1994 under the designation pWDC-7 with the AgriculturalResearch Culture Collection and given the deposit designation NRRL#21339N.

[0077] AraPT1Pro containing the Arabidopsis protox-1 promoter wasdeposited Dec. 15, 1995, as pWDC-11 (NRRL#B-21515)

[0078] A plasmid containing the maize protox-1 promoter fused to theremainder of the maize protox-1 coding sequence was deposited Mar. 19,1996 as pWDC-14 (NRRL #B-21546).

[0079] A plasmid containing the sugar beet protox-1 promoter wasdeposited Dec. 6, 1996, as pWDC-20 (NRRL #B-21650).

DEFINITIONS

[0080] For clarity, certain terms used in the specification are definedand presented as follows:

[0081] Associated With/Operatively Linked: refers to two DNA sequencesthat are related physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operativelylinked, or situated such that the regulator DNA sequence will affect theexpression level of the coding or structural DNA sequence.

[0082] Chimeric Gene: a recombinant DNA sequence in which a promoter orregulatory DNA sequence is operatively linked to, or associated with, aDNA sequence that codes for an mRNA or which is expressed as a protein,such that the regulator DNA sequence is able to regulate transcriptionor expression of the associated DNA sequence. The regulator DNA sequenceof the chimeric gene is not normally operatively linked to theassociated DNA sequence as found in nature.

[0083] Coding DNA Sequence: a DNA sequence that is translated in anorganism to produce a protein.

[0084] Corresponding To: in the context of the present invention,“corresponding to” means that when the amino acid sequences of variousprotox enzymes are aligned with each other, such as in Table 1A, theamino acids that “correspond to” certain enumerated positions in Table1A are those that align with these positions in Table 1A, but that arenot necessarily in these exact numerical positions relative to theparticular protox enzyme's amino acid sequence. Likewise, when the aminoacid sequence of a particular protox enzyme (for example, the soybeanprotox enzyme) is aligned with the amino acid sequence of a referenceprotox enzyme (for example, the Arabidopsis protox-1 sequence given inSEQ ID NO:2), the amino acids in the soybean protox sequence that“correspond to” certain enumerated positions of SEQ ID NO:2 are thosethat align with these positions of SEQ ID NO:2, but are not necessarilyin these exact numerical positions of the soybean protox enzyme's aminoacid sequence.

[0085] DNA Shuffling: DNA shuffling is a method to introduce mutationsor rearrangements, preferably randomly, in a DNA molecule or a method togenerate exchanges of DNA sequences between two or more DNA molecules,preferably randomly. The DNA molecule resulting from DNA shuffling is a“shuffled DNA molecule,” that is a non-naturally occurring DNA moleculederived from at least one template DNA molecule. The shuffled DNAencodes an enzyme modified with respect to the enzyme encoded by thetemplate DNA, and preferably has an altered biological activity withrespect to the enzyme encoded by the template DNA.

[0086] Herbicide: a chemical substance used to kill or suppress thegrowth of plants, plant cells, plant seeds, or plant tissues.

[0087] Heterologous DNA Sequence: a DNA sequence not naturallyassociated with a host cell into which it is introduced, includingnon-naturally occurring multiple copies of a naturally occurring DNAsequence.

[0088] Homologous DNA Sequence: a DNA sequence naturally associated witha host cell into which it is introduced.

[0089] Homoplasmic: refers to a plant, plant tissue or plant cell,wherein all of the plastids are genetically identical. In differenttissues or stages of development, the plastids may take different forms,e.g., chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts,and so forth.

[0090] Inhibitor: a chemical substance that inactivates the enzymaticactivity of a protein such as a biosynthetic enzyme, receptor, signaltransduction protein, structural gene product, or transport protein thatis essential to the growth or survival of the plant. In the context ofthe instant invention, an inhibitor is a chemical substance thatinactivates the enzymatic activity of protox. The term “herbicide” isused herein to define an inhibitor when applied to plants, plant cells,plant seeds, or plant tissues.

[0091] Isolated: in the context of the present invention, an isolatednucleic acid molecule or an isolated enzyme is a nucleic acid moleculeor enzyme that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatednucleic acid molecule or enzyme may exist in a purified form or mayexist in a non-native environment such as, for example, a transgenichost cell.

[0092] Minimal Promoter: promoter elements, particularly a TATA element,that are inactive or that have greatly reduced promoter activity in theabsence of upstream activation. In the presence of a suitabletranscrption factor, the minimal promoter functions to permittranscription.

[0093] Modified Enzyme Activity: enzyme activity different from thatwhich naturally occurs in a plant (i.e. enzyme activity that occursnaturally in the absence of direct or indirect manipulation of suchactivity by man), which is tolerant to inhibitors that inhibit thenaturally occurring enzyme activity.

[0094] Nucleic Acid Molecule: a linear segment of single- ordouble-stranded DNA or RNA that can be isolated from any source. In thecontext of the present invention, the nucleic acid molecule ispreferably a segment of DNA.

[0095] Plant: refers to any plant or part of a plant at any stage ofdevelopment. Therein are also included cuttings, cell or tissue culturesand seeds. As used in conjunction with the present invention, the term“plant tissue” includes, but is not limited to, whole plants, plantcells, plant organs, plant seeds, protoplasts, callus, cell cultures,and any groups of plant cells organized into structural and/orfunctional units.

[0096] Plastome: the genome of a plastid.

[0097] Protox-1: chloroplast protox.

[0098] Protox-2: mitochondrial protox.

[0099] Significant Increase: an increase in enzymatic activity that islarger than the margin of error inherent in the measurement technique,preferably an increase by about 2-fold or greater of the activity of thewild-type enzyme in the presence of the inhibitor, more preferably anincrease by about 5-fold or greater, and most preferably an increase byabout 10-fold or greater.

[0100] Substantially Similar: with respect to nucleic acids, a nucleicacid molecule that has at least 60 percent sequence identity with areference nucleic acid molecule. In a preferred embodiment, asubstantially similar DNA sequence is at least 80% identical to areference DNA sequence; in a more preferred embodiment, a substantiallysimilar DNA sequence is at least 90% identical to a reference DNAsequence; and in a most preferred embodiment, a substantially similarDNA sequence is at least 95% identical to a reference DNA sequence. Asubstantially similar nucleotide sequence typically hybridizes to areference nucleic acid molecule, or fragments thereof, under thefollowing conditions: hybridization at 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C.; wash with 2×SSC, 1% SDS, at 50°C. With respect to proteins or peptides, a substantially similar aminoacid sequence is an amino acid sequence that is at least 90% identicalto the amino acid sequence of a reference protein or peptide and hassubstantially the same activity as the reference protein or peptide.

[0101] Tolerance/Resistance: the ability to continue normal growth orfunction when exposed to an inhibitor or herbicide.

[0102] Transformation: a process for introducing heterologous DNA into acell, tissue, or plant. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof.

[0103] Transit Peptide: a signal polypeptide that is translated inconjunction with a protein encoded by a DNA molecule, forming apolypeptide precursor. In the process of transport to a selected sitewithin the cell, a chloroplast for example, the transit peptide can becleaved from the remainder of the polypeptide precursor to provide anactive or mature protein.

[0104] Transformed: refers to an organism such as a plant into which aheterologous DNA molecule has been introduced. The DNA molecule can bestably integrated into the genome of the plant, wherein the genome ofthe plant encompasses the nuclear genome, the plastid genome and themitochondrial genome. In a transformed plant, the DNA molecule can alsobe present as an extrachromosomal molecule. Such an extrachromosomalmolecule can be auto-replicating. A “non-transformed” plant refers to awild-type organism, i.e., a plant, which does not contain theheterologous DNA molecule.

[0105] Transplastome: a transformed plastid genome.

[0106] Nucleotides are indicated by their bases by the followingstandard abbreviations: adenine (A), cytosine (C), thyrnine (T), andguanine (G). Amino acids are likewise indicated by the followingstandard abbreviations: alanine (ala; A), arginine (Arg; R), asparagine(Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q),glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine(Ile; I), leucine (Leu; L), lysine (lys; K), methionine (Met; M),phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).Furthermore (Xaa; X) represents any amino acid.

DETAILED DESCRIPTION OF THE INVENTION

[0107] I. Plant Protox Coding Sequences

[0108] In one aspect, the present invention is directed to an isolatedDNA molecule that encodes protoporphyrinogen oxidase (referred to hereinas “protox”), the enzyme that catalyzes the oxidation ofprotoporphyrinogen IX to protoporphyrin IX, from wheat, soybean, cotton,sugar beet, oilseed rape, rice, sorghum, and sugar cane. The partial DNAcoding sequence and corresponding amino acid sequence for a wheat protoxenzyme are provided as SEQ ID NOs:9 and 10, respectively. The DNA codingsequence and corresponding amino acid sequence for a soybean protoxenzyme are provided as SEQ ID NOs:11 and 12, respectively. The DNAcoding sequence and corresponding amino acid sequence for a cottonprotox enzyme are provided as SEQ ID NOs:15 and 16, respectively. TheDNA coding sequence and corresponding amino acid sequence for a sugarbeet protox enzyme are provided as SEQ ID NOs:17 and 18, respectively.The DNA coding sequence and corresponding amino acid sequence for anoilseed rape protox enzyme are provided as SEQ ID NOs:19 and 20,respectively. The partial DNA coding sequence and corresponding aminoacid sequence for a rice protox enzyme are provided as SEQ ID NOs:21 and22, respectively. The partial DNA coding sequence and correspondingamino acid sequence for a sorghum protox enzyme are provided as SEQ IDNOs:23 and 24, respectively. The partial DNA coding sequence andcorresponding amino acid sequence for a sugar cane protox enzyme areprovided as SEQ ID NOs:36 and 37, respectively.

[0109] The DNA coding sequences and corresponding amino acid sequencesfor protox enzymes from Arabidopsis thaliana and maize are providedherein as SEQ ID NOs:1-4 (Arabidopsis) and SEQ ID NOs:5-8 (maize).

[0110] The invention therefore is directed to a DNA molecule encoding aprotoporphyrinogen oxidase (protox) comprising a eukaryotic protoxselected from the group consisting of a wheat protox enzyme, a soybeanprotox enzyme, a cotton protox enzyme, a sugar beet protox enzyme, anoilseed rape protox enzyme, a rice protox enzyme, a sorghum protoxenzyme, and a sugar cane protox enzyme.

[0111] Preferred within the scope of the invention are isolated DNAmolecules encoding the protoporphyrinogen oxidase (protox) enzyme fromdicotyledonous plants, but especially from soybean plants, cottonplants, sugar beet plants and oilseed rape plants, such as those givenin SEQ ID NOS:11, 15, 17 and 19. More preferred are isolated DNAmolecules encoding the protoporphyrinogen oxidase (protox) enzyme fromsoybean, such as given in SEQ ID NO:11, and sugar beet, such as given inSEQ ID NO:17.

[0112] Also preferred are isolated DNA molecules encoding theprotoporphyrinogen oxidase (protox) enzyme from monocotyledonous plants,but especially from wheat plants, rice plants, sorghum plants, and sugarcane plants, such as those given in SEQ ID NOS:9, 21, 23, and 36. Morepreferred are isolated DNA molecules encoding the protoporphyrinogenoxidase (protox) enzyme from wheat such as given in SEQ ID NO:9.

[0113] In another aspect, the present invention is directed to isolatedDNA molecules encoding the protoporphyrinogen oxidase (protox) enzymeprotein from a dicotyledonous plant, wherein the protein comprises theamino acid sequence selected from the group consisting of SEQ ID NOs:12,16, 18 and 20. Further comprised are isolated DNA molecules encoding theprotoporphyrinogen oxidase (protox) enzyme protein from amonocotyledonous plant, wherein the protein comprises the amino acidsequence selected from the group consisting of SEQ ID NOs:10, 22, 24,and 37. More preferred is an isolated DNA molecule encoding theprotoporphyrinogen oxidase (protox) enzyme wherein the protein comprisesthe amino acid sequence from wheat such as given in SEQ ID NO:10. Morepreferred is an isolated DNA molecule encoding the protoporphyrinogenoxidase (protox) enzyme wherein the protein comprises the amino acidsequence from soybean, such as given in SEQ ID NO:12 and sugar beet,such as given in SEQ ID NO:18.

[0114] Using the information provided by the present invention, the DNAcoding sequence for the protoporphyrinogen oxidase (protox) enzyme fromany eukaryotic organism may be obtained using standard methods.

[0115] In another aspect, the present invention is directed to anisolated DNA molecule that encodes a wheat protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:9 under the following hybridization and washconditions:

[0116] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0117] (b) wash in 2×SSC, 1% SDS at 50° C.

[0118] In yet another aspect, the present invention is directed to anisolated DNA molecule that encodes a soybean protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:11 under the following hybridization and washconditions:

[0119] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, I mM EDTA at 50° C.; and

[0120] (b) wash in 2×SSC, 1% SDS at 50° C.

[0121] In still another aspect, the present invention is directed to anisolated DNA molecule that encodes a cotton protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:15 under the following hybridization and washconditions:

[0122] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0123] (b) wash in 2×SSC, l% SDS at 50° C.

[0124] In another aspect, the present invention is directed to anisolated DNA molecule that encodes a sugar beet protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:17 under the following hybridization and washconditions:

[0125] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0126] (b) wash in 2×SSC, 1% SDS at 50° C.

[0127] In another aspect, the present invention is directed to anisolated DNA molecule that encodes an oilseed rape protox enzyme andthat comprises a nucleotide sequence that hybridizes to the codingsequence shown in SEQ ID NO:19 under the following hybridization andwash conditions:

[0128] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0129] (b) wash in 2×SSC, 1% SDS at 50° C.

[0130] In another aspect, the present invention is directed to anisolated DNA molecule that encodes a rice protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:21 under the following hybridization and washconditions:

[0131] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0132] (b) wash in 2×SSC, 1% SDS at 50° C.

[0133] In another aspect, the present invention is directed to anisolated DNA molecule that encodes a sorghum protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:23 under the following hybridization and washconditions:

[0134] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0135] (b) wash in 2×SSC, 1% SDS at 50° C.

[0136] In another aspect, the present invention is directed to anisolated DNA molecule that encodes a sugar cane protox enzyme and thatcomprises a nucleotide sequence that hybridizes to the coding sequenceshown in SEQ ID NO:36 under the following hybridization and washconditions:

[0137] (a) hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄pH 7.0, 1 mM EDTA at 50° C.; and

[0138] (b) wash in 2×SSC, 1% SDS at 50° C.

[0139] The isolated eukaryotic protox sequences taught by the presentinvention may be manipulated according to standard genetic engineeringtechniques to suit any desired purpose. For example, the entire protoxsequence or portions thereof may be used as probes capable ofspecifically hybridizing to protox coding sequences and messenger RNA's.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among protox coding sequencesand are preferably at least 10 nucleotides in length, and mostpreferably at least 20 nucleotides in length. Such probes may be used toamplify and analyze protox coding sequences from a chosen organism viathe well known process of polymerase chain reaction (PCR). Thistechnique may be useful to isolate additional protox coding sequencesfrom a desired organism or as a diagnostic assay to determine thepresence of protox coding sequences in an organism.

[0140] Factors that affect the stability of hybrids determine thestringency of the hybridization. One such factor is the meltingtemperature T_(m), which can be easily calculated according to theformula provided in DNA PROBES, George H. Keller and Mark M. Manak,Macmillan Publishers Ltd, 1993, Section one: Molecular HybridizationTechnology; page 8 ff. The preferred hybridization temperature is in therange of about 25° C. below the calculated melting temperature T_(m) andpreferably in the range of about 12-15° C. below the calculated meltingtemperature T_(m) and in the case of oligonucleotides in the range ofabout 5-10° C. below the melting temperature T_(m).

[0141] Comprised by the present invention are DNA molecules thathybridize to a DNA molecule according to the invention as definedhereinbefore, but preferably to an oligonucleotide probe obtainable fromthe DNA molecule comprising a contiguous portion of the sequence of theprotoporphyrinogen oxidase (protox) enzyme at least 10 nucleotides inlength, under moderately stringent conditions.

[0142] The invention further embodies the use of a nucleotide probecapable of specifically hybridizing to a plant protox gene or mRNA of atleast 10 nucleotides length in a polymerase chain reaction (PCR).

[0143] In a further embodiment, the present invention provides probescapable of specifically hybridizing to a eukaryotic DNA sequenceencoding a protoporphyrinogen oxidase activity or to the respective mRNAand methods for detecting the DNA sequences in eukaryotic organismsusing the probes according to the invention.

[0144] Protox specific hybridization probes may also be used to map thelocation of the native eukaryotic protox gene(s) in the genome of achosen organism using standard techniques based on the selectivehybridization of the probe to genomic protox sequences. These techniquesinclude, but are not limited to, identification of DNA polymorphismsidentified or contained within the protox probe sequence, and use ofsuch polymorphisms to follow segregation of the protox gene relative toother markers of known map position in a mapping population derived fromself fertilization of a hybrid of two polymorphic parental lines (seee.g. Helentjaris et al., Plant Mol. Biol. 5: 109 (1985). Sommer et al.Biotechniques 12: 82 (1992); D'Ovidio et al., Plant Mol. Biol. 15: 169(1990)). While any eukaryotic protox sequence is contemplated to beuseful as a probe for mapping protox genes from any eukaryotic organism,preferred probes are those protox sequences from organisms more closelyrelated to the chosen organism, and most preferred probes are thoseprotox sequences from the chosen organism. Mapping of protox genes inthis manner is contemplated to be particularly useful in plants forbreeding purposes. For instance, by knowing the genetic map position ofa mutant protox gene that confers herbicide resistance, flanking DNAmarkers can be identified from a reference genetic map (see, e.g.,Helentjaris, Trends Genet. 3: 217 (1987)). During introgression of theherbicide resistance trait into a new breeding line, these markers canthen be used to monitor the extent of protox-linked flanking chromosomalDNA still present in the recurrent parent after each round ofback-crossing.

[0145] Protox specific hybridization probes may also be used toquantitate levels of protox mRNA in an organism using standardtechniques such as Northern blot analysis. This technique may be usefulas a diagnostic assay to detect altered levels of protox expression thatmay be associated with particular adverse conditions such as autosomaldominant disorder in humans characterized by both neuropsychiatricsymptoms and skin lesions, which are associated with decreased levels ofprotox activity (Brenner and Bloomer, New Engl. J. Med. 302: 765(1980)).

[0146] A further embodiment of the invention is a method of producing aDNA molecule comprising a DNA portion encoding a protein havingprotoporphyrinogen oxidase (protox) enzyme activity comprising:

[0147] (a) preparing a nucleotide probe capable of specificallyhybridizing to a plant protox gene or mRNA, wherein the probe comprisesa contiguous portion of the coding sequence for a protox protein from aplant of at least 10 nucleotides length;

[0148] (b) probing for other protox coding sequences in populations ofcloned genomic DNA fragments or cDNA fragments from a chosen organismusing the nucleotide probe prepared according to step (a); and

[0149] (c) isolating and multiplying a DNA molecule comprising a DNAportion encoding a protein having protoporphyrinogen oxidase (protox)enzyme activity.

[0150] A further embodiment of the invention is a method of isolating aDNA molecule from any plant comprising a DNA portion encoding a proteinhaving protoporphyrinogen oxidase (protox) enzyme activity.

[0151] (a) preparing a nucleotide probe capable of specificallyhybridizing to a plant protox gene or mRNA, wherein the probe comprisesa contiguous portion of the coding sequence for a protox protein from aplant of at least 10 nucleotides length;

[0152] (b) probing for other protox coding sequences in populations ofcloned genomic DNA fragments or cDNA fragments from a chosen organismusing the nucleotide probe prepared according to step (a); and

[0153] (c) isolating a DNA molecule comprising a DNA portion encoding aprotein having protoporphyrinogen oxidase (protox) enzyme activity.

[0154] The invention further comprises a method of producing anessentially pure DNA sequence coding for a protein exhibitingprotoporphyrinogen oxidase (protox) enzyme activity, which methodcomprises:

[0155] (a) preparing a genomic or a cDNA library from a suitable sourceorganism using an appropriate cloning vector;

[0156] (b) hybridizing the library with a probe molecule; and

[0157] (c) identifying positive hybridizations of the probe to the DNAclones from the library that is clones potentially containing thenucleotide sequence corresponding to the amino acid sequence forprotoporphyrinogen oxidase (protox).

[0158] The invention further comprises a method of producing anessentially pure DNA sequence coding for a protein exhibitingprotoporphyrinogen oxidase (protox) enzyme activity, which methodcomprises:

[0159] (a) preparing total DNA from a genomic or a cDNA library;

[0160] (b) using the DNA of step (a) as a template for PCR reaction withprimers representing low degeneracy portions of the amino acid sequenceof protoporphyrinogen oxidase (protox).

[0161] A further object of the invention is an assay to identifyinhibitors of protoporphyrinogen oxidase (protox) enzyme activity thatcomprises:

[0162] (a) incubating a first sample of protoporphyrinogen oxidase(protox) and its substrate;

[0163] (b) measuring an uninhibited reactivity of the protoporphyrinogenoxidase (protox) from step (a);

[0164] (c) incubating a first sample of protoporphyrinogen oxidase(protox) and its substrate in the presence of a second sample comprisingan inhibitor compound;

[0165] (d) measuring an inhibited reactivity of the protoporphyrinogenoxidase (protox) enzyme from step (c); and

[0166] (e) comparing the inhibited reactivity to the uninhibitedreactivity of protoporphyrinogen oxidase (protox) enzyme.

[0167] A further object of the invention is an assay to identifyinhibitor-resistant protoporphyrinogen oxidase (protox) mutants thatcomprises:

[0168] (a) incubating a first sample of protoporphyrinogen oxidase(protox) enzyme and its substrate in the presence of a second samplecomprising a protoporphyrinogen oxidase (protox) enzyme inhibitor;

[0169] (b) measuring an unmutated reactivity of the protoporphyrinogenoxidase (protox) enzyme from step (a);

[0170] (c) incubating a first sample of a mutated protoporphyrinogenoxidase (protox) enzyme and its substrate in the presence of a secondsample comprising protoporphyrinogen oxidase (protox) enzyme inhibitor;

[0171] (d) measuring a mutated reactivity of the mutatedprotoporphyrinogen oxidase (protox) enzyme from step (c); and

[0172] (e) comparing the mutated reactivity to the unmutated reactivityof the protoporphyrinogen oxidase (protox) enzyme.

[0173] A further object of the invention is a protox enzyme inhibitorobtained by a method according to the invention.

[0174] For recombinant production of the enzyme in a host organism, theprotox coding sequence may be inserted into an expression cassettedesigned for the chosen host and introduced into the host where it isrecombinantly produced. The choice of specific regulatory sequences suchas promoter, signal sequence, 5′ and 3′ untranslated sequences, andenhancer, is within the level of skill of the routineer in the art. Theresultant molecule, containing the individual elements linked in properreading frame, may be inserted into a vector capable of beingtransformed into the host cell. Suitable expression vectors and methodsfor recombinant production of proteins are well known for host organismssuch as E. coli (see, e.g. Studier and Moffatt, J. Mol. Biol. 189: 113(1986); Brosius, DNA 8: 759 (1989)), yeast (see, e.g., Schneider andGuarente, Meth. Enzymol. 194: 373 (1991)) and insect cells (see, e.g.,Luckow and Summers, Bio/Technol. 6: 47 (1988)). Specific examplesinclude plasmids such as pBluescript (Stratagene, La Jolla, Calif.),pFLAG (International Biotechnologies, Inc., New Haven, Conn.), pTrcHis(Invitrogen, La Jolla, Calif.), and baculovirus expression vectors,e.g., those derived from the genome of Autographica californica nuclearpolyhedrosis virus (AcMNPV). A preferred baculovirus/insect system ispV111392/Sf21 cells (Invitrogen, La Jolla, Calif.).

[0175] Recombinantly produced eukaryotic protox enzyme is useful for avariety of purposes. For example, it may be used to supply protoxenzymatic activity in vitro. It may also be used in an in vitro assay toscreen known herbicidal chemicals whose target has not been identifiedto determine if they inhibit protox. Such an in vitro assay may also beused as a more general screen to identify chemicals that inhibit protoxactivity and that are therefore herbicide candidates. Recombinantlyproduced eukaryotic protox enzyme may also be used in an assay toidentify inhibitor-resistant protox mutants (see Internationalapplication no. PCT/IB95/00452 filed Jun. 8, 1995, published Dec. 21,1995 as WO 95/34659, incorporated by reference herein in its entirety).Alternatively, recombinantly produced protox enzyme may be used tofurther characterize its association with known inhibitors in order torationally design new inhibitory herbicides as well as herbicidetolerant forms of the enzyme.

[0176] II. Inhibitor Resistant Plant Protox Enzymes

[0177] In another aspect, the present invention teaches modificationsthat can be made to the amino acid sequence of any eukaryoticprotoporphyrinogen oxidase (referred to herein as “protox”) enzyme toyield an inhibitor-resistant form of this enzyme. Preferably, theeukaryotic protox enzyme is a plant protox enzyme. The present inventionis directed to inhibitor-resistant protox enzymes having themodifications taught herein, to DNA molecules encoding these modifiedenzymes, and to chimeric genes capable of expressing these modifiedenzymes in plants.

[0178] The present invention is thus directed to an isolated DNAmolecule encoding a modified eukaryotic protoporphyrinogen oxidase(protox) having at least one amino acid modification, wherein the aminoacid modification has the property of conferring resistance to a protoxinhibitor, that is wherein the modified protox is tolerant to aninhibitor in amounts that inhibit the naturally occurring eukaryoticprotox. As used herein “inhibit” refers to a reduction in enzymaticactivity observed in the presence of a subject compound compared to thelevel of activity observed in the absence of the subject compound,wherein the percent level of reduction is preferably at least 10%, morepreferably at least 50%, and most preferably at least 90%.

[0179] Preferred is a DNA molecule encoding a modified eukaryoticprotoporphyrinogen oxidase (protox) that is a plant protox, wherein themodified protox is tolerant to a herbicide in amounts that inhibit thenaturally occurring protox activity. Even more preferred is a protoxselected from the group consisting of an Arabidopsis protox enzyme, amaize protox enzyme, a wheat protox enzyme, a soybean protox enzyme, acotton protox enzyme, a sugar beet protox enzyme, an oilseed rape protoxenzyme, a rice protox enzyme, a sorghum protox enzyme, and a sugar caneprotox enzyme having at least one amino acid modification, wherein themodified protox is tolerant to a herbicide in amounts that inhibit thenaturally occurring protox activity.

[0180] As used herein, the expression “substantially conserved aminoacid sequences” refers to regions of amino acid homology betweenpolypeptides comprising protox enzymes from different sources. In thepresent invention, seventeen substantially conserved amino acidsub-sequences, designated 1-17 respectively, are shown in Table 1B. Oneskilled in the art could align the amino acid sequences of protoxenzymes from different sources, as has been done in Table 1A, toidentify the sub-sequences therein that make up the substantiallyconserved amino acid sequences defined herein. Stated another way, agiven sub-sequence from one source “corresponds to” a homologoussubsequence from a different source. The skilled person could thendetermine whether the identified sub-sequences have the characteristicsdisclosed and claimed in the present application.

[0181] Therefore, a preferred embodiment of the present invention isdirected to a nucleic acid molecule comprising a nucleotide sequenceisolated from a plant that encodes an enzyme having protoporphyrinogenoxidase (protox) activity, wherein the nucleic acid molecule is capableof being incorporated into a nucleic acid construct used to transform aplant containing wild-type, herbicide-sensitive protox, wherein thenucleotide sequence has at least one point mutation relative to awild-type nucleotide sequence encoding plant protox, such that upontransformation with the nucleic acid construct the plant is renderedherbicide-tolerant.

[0182] More particularly, a preferred embodiment of the presentinvention is directed to a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises at least one of thefollowing amino acid sub-sequences:

[0183] (a) ΔPΔ₁F, wherein Δ₁ is an amino acid other than arginine;

[0184] (b) FΔ₂S, wherein Δ₂ is an amino acid other than cysteine;

[0185] (c) YΔ₃G, wherein Δ₃ is an amino acid other than alanine;

[0186] (d) AΔ₄D, wherein Δ₄ is an amino acid other than glycine;

[0187] (e) YΔ₅P, wherein Δ₅ is an amino acid other than proline;

[0188] (f) PΔ₆A, wherein Δ₆ is an amino acid other than valine;

[0189] (g) Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine;

[0190] (h) YIGGΔ₈, wherein Δ₈ is an amino acid other than alanine orserine;

[0191] (i) AΔ₉P, wherein Δ₉ is an amino acid other than isoleucine; and

[0192] (j) GΔ₁₀A, wherein Δ₁₀ is an amino acid other than valine (Table1B; sub-sequences 1-10).

[0193] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence APΔ₁F, wherein Δ₁ is an amino acid other than arginine.Most preferably, Δ₁ is cysteine.

[0194] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence FΔ₂S, wherein Δ₂ is an amino acid other than cysteine. Mostpreferably, Δ₂ is phenylalanine, leucine, or lysine.

[0195] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine. Mostpreferably, Δ₃ is valine, threonine, leucine, cysteine, or isoleucine.

[0196] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence AΔ₄D, wherein Δ₄ is an amino acid other than glycine. Mostpreferably, Δ₄ is serine or leucine.

[0197] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence Δ₅ is an amino acid other than proline. Most preferably, Δ₅is serine or histidine.

[0198] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence PΔ₆A, wherein Δ₆ is an amino acid other than valine. Mostpreferably, Δ₆ is leucine.

[0199] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine. Mostpreferably, Δ₇ is cysteine, isoleucine, leucine, threonine, methionine,valine, alanine, or arginine.

[0200] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence FΔ₂S, wherein Δ₈ is an amino acid other than alanine orserine. Most preferably, Δ₈ is proline.

[0201] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence AΔGP, wherein Δ₉ is an amino acid other than isoleucine.Most preferably, Δ₉ is threonine, histidine, glycine, or asparagine.

[0202] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence GΔ₁₀A, wherein Δ₁₀ is an amino acid other than valine. Mostpreferably, Δ₁₀ is alanine.

[0203] Another preferred embodiment of the present invention is directedto a nucleic acid molecule comprising a nucleotide sequence isolatedfrom a plant that encodes a modified enzyme having protoporphyrinogenoxidase (protox) activity, wherein the modified enzyme is resistant toan inhibitor of a naturally occurring protox enzyme, wherein themodified enzyme comprises at least one of the following amino acidsub-sequences:

[0204] (a) APΔ₁F, wherein Δ₁ is an amino acid other than arginine;

[0205] (b) FΔ₂S, wherein Δ₂ is an amino acid other than cysteine;

[0206] (c) YΔ₃G, wherein Δ₃ is an amino acid other than alanine;

[0207] (d) AΔ₄D, wherein Δ₄ is an amino acid other than glycine;

[0208] (e) YΔ₅P, wherein Δ₅ is an amino acid other than proline;

[0209] (f) PΔ₆A, wherein Δ₆ is an amino acid other than valine;

[0210] (g) Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine;

[0211] (h) YIGGΔ₈, wherein Δ₈ is an amino acid other than alanine orserine;

[0212] (i) AΔ₉P, wherein Δ₉ is an amino acid other than isoleucine; and

[0213] (j) GΔ₁₀A, wherein Δ₁₀ is an amino acid other than valine

[0214] (Table 1B; sub-sequences 1-10), and wherein the modified enzymefurther comprises at least one additional amino acid sub-sequenceselected from the group consisting of:

[0215] (k) QΔ₁₁S, wherein Δ₁₁ is an amino acid other than proline;

[0216] (l) IGGΔ₁₂, wherein Δ₁₂ is an amino acid other than threonine;

[0217] (m) SWXLΔ₁₃, wherein Δ₁₃ is an amino acid other than serine;

[0218] (n) LΔ₁₄Y, wherein Δ₁₄ is an amino acid other than asparagine;and

[0219] (o) GΔ₁₅XGL, wherein Δ₁₅ is an amino acid other than tyrosine.

[0220] Preferred is a nucleic acid molecule comprising a nucleotidesequence isolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the modified enzyme comprises the amino acidsub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine, orthe amino acid sub-sequence Δ₇IG, wherein Δ₇ is an amino acid other thantyrosine, and wherein the modified enzyme further comprises at least oneadditional amino acid sub-sequence selected from the group consistingof:

[0221] (k) QΔ₁₁S, wherein Δ₁₁ is an amino acid other than proline;

[0222] (l) IGGΔ₁₂, wherein Δ₁₂ is an amino acid other than threonine;

[0223] (m) SWXLΔ₁₃, wherein Δ₁₃ is an amino acid other than serine;

[0224] (n) LΔ₁₄Y, wherein Δ₁₄ is an amino acid other than asparagine;and

[0225] (o) GΔ₁₅XGL, wherein Δ₁₅ is an amino acid other than tyrosine.

[0226] Preferably, Δ₁₁l is leucine, Δ₁₂ is isoleucine or alanine, Δ₁₃ isleucine, Δ₁₄ is serine, and Δ₁₅ is cysteine.

[0227] Another preferred embodiment of the present invention is directedto a nucleic acid molecule comprising a nucleotide sequence isolatedfrom a plant that encodes a modified enzyme having protoporphyrinogenoxidase (protox) activity, wherein the modified enzyme is resistant toan inhibitor of a naturally occurring protox enzyme, wherein themodified enzyme comprises: the amino acid sub-sequence Δ₇IG, wherein Δ₇is an amino acid other than tyrosine; the amino acid sub-sequencesIGGΔ₁₂, wherein Δ₁₂ is an amino acid other than threonine; and the aminoacid sub-sequence SWXLΔ₁₃, wherein Δ₁₃ is an amino acid other thanserine. Most preferably, Δ₇ is isoleucine, Δ₁₂ is isoleucine, and Δ₁₃ isleucine.

[0228] Yet another preferred embodiment of the present invention isdirected to a nucleic acid molecule comprising a nucleotide sequenceisolated from a plant that encodes a modified enzyme havingprotoporphyrinogen oxidase (protox) activity, wherein the modifiedenzyme is resistant to an inhibitor of a naturally occurring protoxenzyme, wherein the nucleotide sequence is further characterized in thatat least one of the following conditions is met:

[0229] (a) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence APΔ₁F, wherein Δ₁ is an amino acid other thanarginine;

[0230] (b) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence FΔ₂S, wherein Δ₂ is an amino acid other than cysteine;

[0231] (c) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine;

[0232] (d) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence AΔ₄D, wherein Δ₄ is an amino acid other than glycine;

[0233] (e) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence YΔ₅P, wherein Δ₅ is an amino acid other than proline;

[0234] (f) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence PΔ₆A, wherein Δ₆ is an amino acid other than valine;

[0235] (g) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine;

[0236] (h) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence YIGGΔ_(8,) wherein Δ₈ is an amino acid other thanalanine or serine;

[0237] (i) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence AΔ₉P, wherein Δ₉ is an amino acid other thanisoleucine;

[0238] (j) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence GΔ₁₀A, wherein Δ₁₀ is an amino acid other than valine;

[0239] (k) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine,and the nucleic acid sequence also has a sequence that encodes one ofthe group consisting of:

[0240] (1) sub-sequence QΔ₁₁S, wherein Δ₁₁ is an amino acid other thanproline,

[0241] (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is an amino acid other thanthreonine,

[0242] (3) sub-sequence SWXLΔ₁₃, wherein Δ₁₃ is an amino acid other thanserine,

[0243] (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄ is an amino acid other thanasparagine, and

[0244] (5) sub-sequence GΔ₁₅XGL, wherein Δ₁₅ is an amino acid other thantyrosine;

[0245] (l) the nucleic acid sequence has a sequence that encodes aminoacid sub-sequence Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine,and the nucleic acid sequence also has a sequence that encodes one ofthe group consisting of:

[0246] (1) sub-sequence QΔ₁₁S, wherein Δ₁₁ is an amino acid other thanproline,

[0247] (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is an amino acid other thanthreonine,

[0248] (3) sub-sequence SWXLΔ₁₃, wherein Δ₁₃ is an amino acid other thanserine,

[0249] (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄ is an amino acid other thanasparagine, and

[0250] (5) sub-sequence GΔ₁₅XGL, wherein Δ₁₅ is an amino acid other thantyrosine; and

[0251] (m) the nucleic has a sequence that encodes amino acidsub-sequence TΔ₁₆G, wherein Δ₁₆ is an amino acid other than leucine, andthe nucleic acid sequence also has a sequence that encodes amino acidsub-sequence YVΔ₁₇G, wherein Δ₁₆ is an amino acid other than alanine.

[0252] Preferably, said nucleic acid sequence has a sequence thatencodes amino acid sub-sequence TΔ₁6G, wherein Δ₁₆ is an amino acidother than leucine, and said nucleic acid sequence also has a sequencethat encodes amino acid sub-sequence YVΔ₁₇G, wherein Δ₁₆ is an aminoacid other than alanine.

[0253] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe arginine occurring at the position corresponding to amino acid 88 ofSEQ ID NO:6 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is the DNA moleculewherein the arginine is replaced with a cysteine.

[0254] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe cysteine occurring at the position corresponding to amino acid 159of SEQ ID NO:6 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is the DNA moleculewherein the cysteine is replaced with a phenylalanine or lysine, mostpreferred, wherein the cysteine is replaced with a phenylalanine.

[0255] Also preferred is a DNA encoding a modified protoporphyrinogenoxidase (protox) comprising a plant protox wherein the isoleucineoccurring at the position corresponding to amino acid 419 of SEQ ID NO:6is replaced with another amino acid, wherein the modified protox istolerant to a herbicide in amounts that inhibit the naturally occurringprotox activity. Particularly preferred is a DNA molecule, wherein theisoleucine is replaced with a threonine, histidine, glycine orasparagine most preferred, wherein the isoleucine is replaced with athreonine.

[0256] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe alanine occurring at the position corresponding to amino acid 164 ofSEQ ID NO:6 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the alanine is replaced with a threonine, leucine or valine.

[0257] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe glycine occurring at the position corresponding to amino acid 165 ofSEQ ID NO:6 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the glycine is replaced with a serine or leucine.

[0258] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe tyrosine occurring at the position corresponding to amino acid 370of SEQ ID NO:6 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the tyrosine is replaced with a isoleucine or methionine.

[0259] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe valine occurring at the position corresponding to amino acid 356 ofSEQ ID NO:10 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the valine is replaced with a leucine.

[0260] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe serine occurring at the position corresponding to amino acid 421 ofSEQ ID NO:10 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the serine is replaced with a proline.

[0261] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe valine occurring at the position corresponding to amino acid 502 ofSEQ ID NO:10 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the valine is replaced with a alanine.

[0262] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe alanine occurring at the position corresponding to amino acid 211 ofSEQ ID NO:10 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the alanine is replaced with a valine or threonine.

[0263] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe glycine occurring at the position corresponding to amino acid 212 ofSEQ ID NO:10 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the glycine is replaced with a serine.

[0264] Also preferred is a DNA encoding a modified protoporphyrinogenoxidase (protox) comprising a plant protox wherein the isoleucineoccurring at the position corresponding to amino acid 466 of SEQ IDNO:10 is replaced with another amino acid, wherein the modified protoxis tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the isoleucine is replaced with a threonine.

[0265] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe proline occurring at the position corresponding to amino acid 369 ofSEQ ID NO:12 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the proline is replaced with a serine or histidine.

[0266] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe alanine occurring at the position corresponding to amino acid 226 ofSEQ ID NO:12 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA molecule,wherein the alanine is replaced with a threonine or leucine.

[0267] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe valine occurring at the position corresponding to amino acid 517 ofSEQ ID NO:12 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the valine is replaced with a alanine.

[0268] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe tyrosine occurring at the position corresponding to amino acid 432of SEQ ID NO:12 is replaced with another amino acid, wherein themodified protox is tolerant to a herbicide in amounts that inhibit thenaturally occurring protox activity. Particularly preferred is a DNAmolecule wherein the tyrosine is replaced with a leucine or isoleucine.

[0269] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe proline occurring at the position corresponding to amino acid 365 ofSEQ ID NO:16 is replaced with another amino acid, wherein the modifiedprotox is tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the proline is replaced with a serine.

[0270] Also preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe tyrosine occurring at the position corresponding to amino acid 428of SEQ ID NO:16 is replaced with another amino acid, wherein themodified protox is tolerant to a herbicide in amounts that inhibit thenaturally occurring protox activity. Particularly preferred is a DNAmolecule wherein the tyrosine is replaced with a cysteine or arginine.

[0271] Also preferred is a DNA encoding a modified protoporphyrinogenoxidase (protox) comprising a plant protox wherein the tyrosineoccurring at the position corresponding to amino acid 449 of SEQ IDNO:18 is replaced with another amino acid, wherein the modified protoxis tolerant to a herbicide in amounts that inhibit the naturallyoccurring protox activity. Particularly preferred is a DNA moleculewherein the tyrosine is replaced with a cysteine, leucine, isoleucine,valine or methionine.

[0272] The present invention is further directed to a DNA moleculeencoding a modified protoporphyrinogen oxidase (protox) comprising aplant protox having a first amino acid substitution and a second aminoacid substitution; the first amino acid substitution having the propertyof conferring resistance to a protox inhibitor; and the second aminoacid substitution having the property of enhancing the resistanceconferred by the first amino acid substitution. Preferred is a DNAmolecule encoding a modified protoporphyrinogen oxidase (protox)comprising a plant protox, wherein the plant is selected from the groupconsisting of maize, wheat, soybean, cotton, sugar beet, oilseed rape,rice, sorghum, sugar cane, and Arabidopsis. More preferred is a DNAmolecule encoding a modified protoporphyrinogen oxidase (protox)comprising a plant protox, wherein the plant is selected from the groupconsisting of maize, wheat, soybean, sugar beet, and Arabidopsis.

[0273] Preferred is a DNA molecule wherein the second amino acidsubstitution occurs at a position selected from the group consisting of:

[0274] (a) the position corresponding to the serine at amino acid 305 ofSEQ ID NO:2;

[0275] (b) the position corresponding to the threonine at amino acid 249of SEQ ID NO:2;

[0276] (c) the position corresponding to the proline at amino acid 118of SEQ ID NO:2;

[0277] (d) the position corresponding to the asparagine at amino acid425 of SEQ ID NO:2; and

[0278] (e) the position corresponding to the tyrosine at amino acid 498of SEQ ID NO:2.

[0279] Also preferred is a DNA molecule wherein the first amino acidsubstitution occurs at a position selected from the group consisting of:

[0280] (a) the position corresponding to the arginine at amino acid 88of SEQ ID NO:6;

[0281] (b) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6;

[0282] (c) the position corresponding to the glycine at amino acid 165of SEQ ID NO:6;

[0283] (d) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6;

[0284] (e) the position corresponding to the cysteine at amino acid 159of SEQ ID NO:6;

[0285] (f) the position corresponding to the isoleucine at amino acid419 of SEQ ID NO:6.

[0286] (g) the position corresponding to the valine at amino acid 356 ofSEQ ID NO:10;

[0287] (h) the position corresponding to the serine at amino acid 421 ofSEQ ID NO:10;

[0288] (i) the position corresponding to the valine at amino acid 502 ofSEQ ID NO:10;

[0289] (j) the position corresponding to the alanine at amino acid 211of SEQ ID NO:10;

[0290] (k) the position corresponding to the glycine at amino acid 212of SEQ ID NO:10;

[0291] (l) the position corresponding to the isoleucine at amino acid466 of SEQ ID NO:10;

[0292] (m) the position corresponding to the proline at amino acid 369of SEQ ID NO:12;

[0293] (n) the position corresponding to the alanine at amino acid 226of SEQ ID NO:12;

[0294] (o) the position corresponding to the tyrosine at amino acid 432of SEQ ID NO:12;

[0295] (p) the position corresponding to the valine at amino acid 517 ofSEQ ID NO:12;

[0296] (q) the position corresponding to the tyrosine at amino acid 428of SEQ ID NO:16;

[0297] (r) the position corresponding to the proline at amino acid 365of SEQ ID NO:16; and

[0298] (s) the position corresponding to the tyrosine at amino acid 449of SEQ ID NO:18.

[0299] Particularly preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox whereinthe plant protox comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, and37. Most preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox, whereinthe plant protox comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, and 18.

[0300] More preferred is a DNA molecule, wherein the first amino acidsubstitution occurs at a position selected from the group consisting of:

[0301] (a) the position corresponding to the arginine at amino acid 88of SEQ ID NO:6;

[0302] (b) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6;

[0303] (c) the position corresponding to the glycine at amino acid 165of SEQ ID NO:6;

[0304] (d) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6;

[0305] (e) the position corresponding to the cysteine at amino acid 159of SEQ ID NO:6; and

[0306] (f) the position corresponding to the isoleucine at amino acid419 of SEQ ID NO:6.

[0307] More preferred is a DNA molecule wherein the second amino acidsubstitution occurs at the position corresponding to the serine at aminoacid 305 of SEQ ID NO:2 and the first amino acid substitution occurs ata position selected from the group consisting of:

[0308] (a) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6; and

[0309] (b) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6.

[0310] Particularly preferred is a DNA molecule wherein the serineoccurring at the position corresponding to amino acid 305 of SEQ ID NO:2is replaced with leucine.

[0311] More preferred is a DNA molecule wherein the second amino acidsubstitution occurs at the position corresponding to the threonine atamino acid 249 of SEQ ID NO:2 and the first amino acid substitutionoccurs at a position selected from the group consisting of:

[0312] (a) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6; and

[0313] (b) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6.

[0314] Particularly preferred is a DNA wherein the threonine occurringat the position corresponding to amino acid 249 of SEQ ID NO:2 isreplaced with an amino acid selected from the group consisting ofisoleucine and alanine.

[0315] More preferred is a DNA molecule wherein the second amino acidsubstitution occurs at the position corresponding to the proline atamino acid 118 of SEQ ID NO:2 and the first amino acid substitutionoccurs at a position selected from the group consisting of:

[0316] (a) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6; and

[0317] (b) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6.

[0318] Particularly preferred is a DNA molecule wherein the prolineoccurring at the position corresponding to amino acid 118 of SEQ ID NO:2is replaced with a leucine.

[0319] More preferred is a DNA molecule wherein the second amino acidsubstitution occurs at the position corresponding to the asparagine atamino acid 425 of SEQ ID NO:2 and the first amino acid substitutionoccurs at a position selected from the group consisting of:

[0320] (a) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6; and

[0321] (b) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6.

[0322] Particularly preferred is a DNA molecule wherein the asparagineoccurring at the position corresponding to amino acid 425 of SEQ ID NO:2is replaced with a serine.

[0323] More preferred is a DNA molecule wherein the second amino acidsubstitution occurs the position corresponding to the tyrosine at aminoacid 498 of SEQ ID NO:2 and the first amino acid substitution occurs ata position selected from the group consisting of:

[0324] (a) the position corresponding to the alanine at amino acid 164of SEQ ID NO:6; and

[0325] (b) the position corresponding to the tyrosine at amino acid 370of SEQ ID NO:6.

[0326] Particularly preferred is a DNA molecule wherein the tyrosineoccurring at the position corresponding to amino acid 498 of SEQ ID NO:2is replaced with a cysteine.

[0327] More preferred is a DNA molecule wherein the tyrosine occurringat the position corresponding to amino acid 370 of SEQ ID NO:6 isreplaced with an amino acid selected from the group consisting ofcysteine, isoleucine, leucine, threonine, valine and methionine.

[0328] Particularly preferred is a DNA molecule wherein the tyrosineoccurring at the position corresponding to amino acid 370 of SEQ ID NO:6is replaced with an amino acid selected from the group consisting ofcysteine, isoleucine, leucine, threonine and methionine.

[0329] More preferred is a DNA molecule wherein the alanine occurring atthe position corresponding to residue 164 of SEQ ID NO:6 is replacedwith an amino acid selected from the group consisting of valine,threonine, leucine, cysteine and tyrosine.

[0330] More preferred is a DNA molecule wherein the glycine occurring atthe position corresponding to residue 165 of SEQ ID NO:6 is replacedwith an amino acid selected from the group consisting of serine andleucine.

[0331] Particularly preferred is a DNA molecule wherein the glycineoccurring at the position corresponding to residue 165 of SEQ ID NO:6 isreplaced with a serine.

[0332] Particularly preferred is a DNA molecule wherein the arginineoccurring at the position corresponding to residue 88 of SEQ ID NO:6 isreplaced with a cysteine.

[0333] More preferred is a DNA molecule wherein the cysteine occurringat the position corresponding to residue 159 of SEQ ID NO:6 is replacedwith an amino acid selected from the group consisting of phenylalanineand lysine.

[0334] Particularly preferred is a DNA molecule wherein the cysteineoccurring at the position corresponding to residue 159 of SEQ ID NO:6 isreplaced with a phenylalanine.

[0335] More preferred is a DNA molecule wherein the isoleucine occurringat the position corresponding to residue 419 of SEQ ID NO:6 is replacedwith an amino acid selected from the group consisting of threonine,histidine, glycine and asparagine.

[0336] Particularly preferred is a DNA molecule wherein the isoleucineoccurring at the position corresponding to residue 419 of SEQ ID NO:6 isreplaced with a threonine.

[0337] More preferred is a DNA molecule wherein the valine occurring atthe position corresponding to residue 356 of SEQ ID NO:10 is replacedwith a leucine.

[0338] More preferred is a DNA molecule wherein the serine occurring atthe position corresponding to residue 421 of SEQ ID NO:10 is replacedwith a proline.

[0339] More preferred is a DNA molecule wherein the valine occurring atthe position corresponding to residue 502 of SEQ ID NO:10 is replacedwith a alanine.

[0340] More preferred is a DNA molecule wherein the isoleucine occurringat the position corresponding to residue 466 of SEQ ID NO:10 is replacedwith a threonine.

[0341] More preferred is a DNA molecule wherein the glycine occurring atthe position corresponding to residue 212 of SEQ ID NO:10 is replacedwith a serine.

[0342] More preferred is a DNA molecule wherein the alanine occurring atthe position corresponding to residue 211 of SEQ ID NO:10 is replacedwith a valine or threonine.

[0343] More preferred is a DNA molecule wherein the proline occurring atthe position corresponding to residue 369 of SEQ ID NO:12 is replacedwith a serine or a histidine.

[0344] More preferred is a DNA molecule wherein the alanine occurring atthe position corresponding to residue 226 of SEQ ID NO:12 is replacedwith a leucine or threonine.

[0345] More preferred is a DNA molecule wherein the tyrosine occurringat the position corresponding to residue 432 of SEQ ID NO:12 is replacedwith a leucine or isoleucine.

[0346] More preferred is a DNA molecule wherein the valine occurring atthe position corresponding to residue 517 of SEQ ID NO:12 is replacedwith a alanine.

[0347] More preferred is a DNA molecule wherein the tyrosine occurringat the position corresponding to residue 428 of SEQ ID NO:16 is replacedwith cysteine or arginine.

[0348] More preferred is a DNA molecule wherein the proline occurring atthe position corresponding to residue 365 of SEQ ID NO:16 is replacedwith serine.

[0349] More preferred is a DNA molecule wherein the proline occurring atthe position corresponding to residue 449 of SEQ ID NO:18 is replacedwith an amino acid selected from the group consisting of leucine,isoleucine, valine and methionine.

[0350] The present invention is still further directed to a DNA moleculeencoding a modified protoporphyrinogen oxidase (protox) comprising aplant protox having a double amino acid substitution, wherein both aminoacid substitutions are required for there to be resistance to a protoxinhibitor. Preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox, whereinthe plant is selected from the group consisting of maize, wheat,soybean, cotton, sugar beet, oilseed rape, rice, sorghum, sugar cane,and Arabidopsis. More preferred is a DNA molecule encoding a modifiedprotoporphyrinogen oxidase (protox) comprising a plant protox, whereinthe plant is maize.

[0351] Preferred is a DNA molecule having a double amino acidsubstitution, wherein one amino acid substitution occurs at the positioncorresponding to the leucine at amino acid 347 of SEQ ID NO:6, andwherein the second amino acid substitution occurs at the positioncorresponding to the alanine at amino acid 453 of SEQ ID NO:6.

[0352] Particularly preferred is a DNA molecule having a double aminoacid substitution, wherein a leucine occurring at the positioncorresponding to amino acid 347 of SEQ ID NO:6 is replaced with aserine, and wherein an alanine occurring a the position corresponding toamino acid 453 of SEQ ID NO:6 is replaced with a threonine.

[0353] Inhibitor resistant protox enzymes are also obtained usingmethods involving in vitro recombination, also called DNA shuffling. ByDNA shuffling, mutations, preferably random mutations, are introduced inprotox genes. DNA shuffling also leads to the recombination andrearrangement of sequences within a given protox gene or to therecombination and exchange of sequences between/among two or moredifferent protox genes. These methods allow for the production ofmillions of mutated protox genes. The mutated genes, or “shuffled”genes, are screened for desirable properties, e.g. improved tolerance toherbicides, and for mutations that provide broad spectrum tolerance tothe different classes of inhibitor chemistry. Such screens are describedherein.

[0354] In a preferred embodiment, a mutagenized protox gene is formedfrom at least one template protox gene, wherein the template protox genehas been cleaved into double-stranded-random fragments of a desiredsize. The method of forming the mutagenized protox gene comprising thefollowing steps: adding to the resultant population of double-strandedrandom fragments one or more single or double-stranded oligonucleotides,wherein said oligonucleotides comprise an area of identity and an areaof heterology to the double-stranded template polynucleotide; denaturingthe resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; incubating theresultant population of single-stranded fragments with a polymeraseunder conditions that result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other, thereby forming a mutagenizeddouble-stranded polynucleotide; and repeating the second and third stepsfor at least two further cycles, wherein the resultant mixture in thesecond step of a further cycle includes the mutagenized double-strandedpolynucleotide from the third step of the previous cycle, and thefurther cycle forms a further mutagenized double-strandedpolynucleotide, wherein the mutagenized DNA molecule is a protox genehaving enhanced tolerance to a herbicide that inhibits naturallyoccuring protox activity. In a preferred embodiment, the concentrationof a single species of double-stranded random fragment in the populationof double-stranded random fragments is less than 1% by weight of thetotal DNA. In a further preferred embodiment, the templatedouble-stranded polynucleotide comprises at least about 100 species ofpolynucleotides. In another preferred embodiment, the size of thedouble-stranded random fragments is from about 5-bp to 5-kb. In afurther preferred embodiment, the fourth step of the method comprisesrepeating the second and the third steps for at least 10 cycles. Suchmethod is described, for example, in Stemmer et al., Nature 370: 389-391(1994), in U.S. Pat. No. 5,605,793, and in Crameri et al., Nature 391:288-291 (1998), as well as in WO 97/20078. The aforementioned referencesare all incorporated herein by reference.

[0355] In another preferred embodiment, any combination of two or moredifferent protox genes are mutagenized in vitro by a staggered extensionprocess (StEP), as described, for example, in Zhao et al., NatureBiotechnology 16: 258-261 (1998). The two or more protox genes are usedas template for PCR amplification with the extension cycles of the PCRreaction preferably carried out at a lower temperature than the optimalpolymerization temperature of the polymerase. For example, when athermostable polymerase with an optimal temperature of approximately 72°C. is used, the temperature for the extension reaction is desirablybelow 72° C., more desirably below 65° C., preferably below 60° C., andmore preferably the temperature for the extension reaction is 55° C.Additionally, the duration of the extension reaction of the PCR cyclesis desirably shorter than usually carried out in the art, more desirablyit is less than 30 seconds, preferably it is less than 15 seconds, andmore preferably the duration of the extension reaction is 5 seconds.Only a short DNA fragment is polymerized in each extension reaction,allowing template switch of the extension products between the startingDNA molecules after each cycle of denaturation and annealing, therebygenerating diversity among the extension products. The optimal number ofcycles in the PCR reaction depends on the length of the protox codingregions to be mutagenized, but desirably over 40 cycles, more desirablyover 60 cycles, and preferably over 80 cycles are used. Optimalextension conditions and the optimal number of PCR cycles for everycombination of protox genes are determined as described in usingprocedures well-known in the art. The other parameters for the PCRreaction are essentialy the same as commonly used in the art. Theprimers for the amplification reaction are preferably designed to annealto DNA sequences located outside of the coding sequence of the protoxgenes, for example to DNA sequences of a vector comprising the protoxgenes, whereby the different protox genes used in the PCR reaction arepreferably comprised in separate vectors. The primers desirably annealto sequences located less than 500-bp away from the protox codingsequences, preferably less than 200-bp away from the protox codingsequences, and more preferably less than 120-bp away from the protoxcoding sequences. Preferably, the protox coding sequences are surroundedby restriction sites, which are included in the DNA sequence amplifiedduring the PCR reaction, thereby facilitating the cloning of theamplified products into a suitable vector.

[0356] In another preferred embodiment, fragments of protox genes havingcohesive ends are produced as described in WO 98/05765. The cohesiveends are produced by ligating a first oligonucleotide corresponding to apart of a protox gene to a second oligonucleotide not present in thegene or corresponding to a part of the gene not adjoining to the part ofthe gene corresponding to the first oligonucleotide, wherein the secondoligonucleotide contains at least one ribonucleotide. A double-strandedDNA is produced using the first oligonucleotide as template and thesecond oligonucleotide as primer. The ribonucleotide is cleaved andremoved. The nucleotide(s) located 5′ to the ribonucleotide are alsoremoved, resulting in double-stranded fragments having cohesive ends.Such fragments are randomly reassembled by ligation to obtain novelcombinations of gene sequences.

[0357] Any protox gene or any combination of protox genes may be usedfor in vitro recombination in the context of the present invention. Forexample, a protox gene used is derived from a plant, such as a protoxgene derived from Arabidopsis thaliana, oilseed rape, soybean,sugarbeet, cotton, maize, wheat, rice, sugarcane and sorghum, whosesequences are disclosed herein. Any mutated protox gene is also used, inparticular the mutated protox genes described herein. Since plant protoxgenes exhibit between 70 and 95% identity at the nucleotide level, withthe highest level of identity existing between monocot protox genes,they are readily used in the methods of in vitro recombination describedabove. Protox genes have also been identified from both mammals (i.e.humans and mice) and bacteria (i.e. Bacillus and Myxococcus), and thesegenes are also appropriate for in vitro recombination, although theyexhibit only approximately 40% nucleotide identity. In this case, andwhen such different genes are used, the conditions in the methodsdescribed above are adapted, in particular, the annealing of sequenceswith low homology is favorized, e.g., by addition of polyethyleneglycol, preferably from 0% to 20%, or by addition of salt, preferablyKCl or NaCl, preferably from 10 mM to 100 mM, or by addition of Mg²⁺ions, preferably from 1 mM to 10 mM, or by lowering the annealingtemperature, desirably below 60° C., preferably below 50° C.Additionally, whole protox genes or portions thereof, e.g. specificregions of protox genes, are used in the context of the presentinvention.

[0358] The library of mutated protox genes obtained by the methodsdescribed above are cloned into appropriate expression vectors and theresulting vectors are transformed into an appropriate host, for examplean algae like Chlamydomonas, a yeast or a bacteria. An appropriate hostis preferably a host that otherwise lacks protox gene activity, forexample E. coli strain SASX38 (Sasarman et al. (1979), J. Gen.Microbiol. 113: 297). Host cells transformed with the vectors comprisingthe library of mutated protox genes are cultured on medium that containsinhibitory concentrations of the inhibitor, and those colonies that growin the presence of the inhibitor are selected. Colonies that grow in thepresence of normally inhibitory concentrations of inhibitor are pickedand purified by repeated restreaking. Their plasmids are purified andthe DNA sequences of cDNA inserts from plasmids that pass this test arethen determined. Any protox inhibitor is used, in particular a protoxinhibitor described herein.

[0359] The present invention is directed to expression cassettes andrecombinant vectors comprising the expression cassettes comprisingessentially a promoter, but especially a promoter that is active in aplant, operatively linked to a DNA molecule encoding theprotoporphyrinogen oxidase (protox) enzyme from a eukaryotic organismaccording to the invention. The expression cassette according to theinvention may in addition further comprise a signal sequence operativelylinked to the DNA molecule, wherein the signal sequence is capable oftargeting the protein encoded by the DNA molecule into the chloroplastor the mitochondria.

[0360] The invention relates to a chimeric gene, which comprises anexpression cassette comprising essentially a promoter, but especially apromoter that is active in a plant, operatively linked to a heterologousDNA molecule encoding a protoporphyrinogen oxidase (protox) enzyme froma eukaryotic organism according to the invention. Preferred is achimeric gene, wherein the DNA molecule encodes an protoporphyrinogenoxidase (protox) enzyme from a plant selected from the group consistingof Arabidopsis, sugar cane, soybean, barley, cotton, tobacco, sugarbeet, oilseed rape, maize, wheat, sorghum, rye, oats, turf and foragegrasses, millet, forage and rice. More preferred is a chimeric gene,wherein the DNA molecule encodes an protoporphyrinogen oxidase (protox)enzyme from a plant selected from the group consisting of soybean,cotton, tobacco, sugar beet, oilseed rape, maize, wheat, sorghum, rye,oats, turf grass, and rice. Particularly preferred is a chimeric gene,wherein the DNA molecule encodes an protoporphyrinogen oxidase (protox)enzyme from a plant selected from the group consisting of wheat,soybean, cotton, sugar beet, oilseed rape, rice and sorghum. Mostpreferred is a chimeric gene, wherein the DNA molecule encodes anprotoporphyrinogen oxidase (protox) enzyme from a plant selected fromthe group consisting of soybean, sugar beet, and wheat.

[0361] More preferred is a chimeric gene comprising a promoter active ina plant operatively linked to a heterologous DNA molecule encoding aprotoporphyrinogen oxidase (protox) selected from the group consistingof a wheat protox comprising the sequence set forth in SEQ ID NO: 10, asoybean protox comprising the sequence set forth in SEQ ID NO:12, cottonprotox comprising the sequence set forth in SEQ ID NO:16, a sugar beetprotox comprising the sequence set forth in SEQ ID NO:18, an oilseedrape protox comprising the sequence set forth in SEQ ID NO:20, a riceprotox comprising the sequence set forth in SEQ ID NO:22, a sorghumprotox comprising the sequence set forth in SEQ ID NO:24, and a sugarcane protox comprising the sequence set forth in SEQ ID NO:37. Morepreferred is a chimeric gene, wherein the protoporphyrinogen oxidase(protox) is selected from the group consisting of a wheat protoxcomprising the sequence set forth in SEQ ID NO:10, a soybean protoxcomprising the sequence set forth in SEQ ID NO:12, and a sugar beetprotox comprising the sequence set forth in SEQ ID NO: 18.

[0362] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from an Arabidopsis species having protox-1activity or protox-2 activity, preferably wherein the protein comprisesthe amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0363] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from maize having protox-1 activity orprotox-2 activity, preferably wherein the protein comprises the aminoacid sequence set forth in set forth in SEQ ID NO:6 or SEQ ID NO:8.

[0364] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from wheat having protox-1 activitypreferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:10.

[0365] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from soybean having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:12.

[0366] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from cotton having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:16.

[0367] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from sugar beet having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:18.

[0368] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from oilseed rape having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:20.

[0369] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from rice having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:22.

[0370] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from sorghum having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:24.

[0371] Particularly preferred is a chimeric gene, wherein the DNAmolecule encodes a protein from sugar cane having protox-1 activity,preferably wherein the protein comprises the amino acid sequence setforth in SEQ ID NO:37.

[0372] The invention also embodies a chimeric gene, which comprises anexpression cassette comprising essentially a promoter, but especially apromoter that is active in a plant, operatively linked to the DNAmolecule encoding an protoporphyrinogen oxidase (protox) enzyme from aeukaryotic organism according to the invention, which is resistant toherbicides at levels that inhibit the corresponding unmodified versionof the enzyme. Preferred is a chimeric gene, wherein the DNA moleculeencodes an protoporphyrinogen oxidase (protox) enzyme from a plantselected from the group consisting of Arabidopsis, sugar cane, soybean,barley, cotton, tobacco, sugar beet, oilseed rape, maize, wheat,sorghum, rye, oats, turf and forage grasses, millet, forage and rice.More preferred is a chimeric gene, wherein the DNA molecule encodes anprotoporphyrinogen oxidase (protox) enzyme from a plant selected fromthe group consisting of soybean, cotton, tobacco, sugar beet, oilseedrape, maize, wheat, sorghum, rye, oats, turf grass, and rice.Particularly preferred is a chimeric gene, wherein the DNA moleculeencodes an protoporphyrinogen oxidase (protox) enzyme from a plantselected from the group consisting of Arabidopsis, soybean, cotton,sugar beet, oilseed rape, maize, wheat, sorghum, and rice.

[0373] Encompassed by the present invention is a chimeric genecomprising a promoter that is active in a plant operatively linked tothe DNA molecule encoding a modified protoporphyrinogen oxidase (protox)comprising a eukaryotic protox having at least one amino acidmodification, wherein the amino acid modification has the property ofconferring resistance to a protox inhibitor.

[0374] Also encompassed by the present invention is a chimeric genecomprising a promoter that is active in a plant operatively linked tothe DNA molecule encoding a modified protoporphyrinogen oxidase (protox)comprising a plant protox having a first amino acid substitution and asecond amino acid substitution; the first amino acid substitution havingthe property of conferring resistance to a protox inhibitor; and thesecond amino acid substitution having the property of enhancing theresistance conferred by the first amino acid substitution. Preferred isthe chimeric gene additionally comprising a signal sequence operativelylinked to the DNA molecule, wherein the signal sequence is capable oftargeting the protein encoded by the DNA molecule into the chloroplastor in the mitochondria.

[0375] The chimeric gene according to the invention may in additionfurther comprise a signal sequence operatively linked to the DNAmolecule, wherein the signal sequence is capable of targeting theprotein encoded by the DNA molecule into the chloroplast. The chimericgene according to the invention may in addition further comprise asignal sequence operatively linked to the DNA molecule, wherein thesignal sequence is capable of targeting the protein encoded by the DNAmolecule into the mitochondria.

[0376] Also encompassed by the present invention is any of the DNAsequences mentioned herein before, which is stably integrated into ahost genome.

[0377] The invention further relates to a recombinant DNA moleculecomprising a plant protoporphyrinogen oxidase (protox) or a functionallyequivalent derivative thereof.

[0378] The invention further relates to a recombinant DNA vectorcomprising the recombinant DNA molecule of the invention.

[0379] A further object of the invention is a recombinant vectorcomprising the chimeric gene according to the invention, wherein thevector is capable of being stably transformed into a host cell.

[0380] A further object of the invention is a recombinant vectorcomprising the chimeric gene according to the invention, wherein thevector is capable of being stably transformed into a plant, plant seeds,plant tissue or plant cell. Preferred is a recombinant vector comprisingthe chimeric gene according to the invention, wherein the vector iscapable of being stably transformed into a plant. The plant, plantseeds, plant tissue or plant cell stably transformed with the vector iscapable of expressing the DNA molecule encoding a protoporphyrinogenoxidase (protox). Preferred is a recombinant vector, wherein the plant,plant seeds, plant tissue or plant cell stably transformed with the thevector is capable of expressing the DNA molecule encoding aprotoporphyrinogen oxidase (protox) from a plant that is resistant toherbicides at levels that inhibit the corresponding unmodified versionof the enzyme.

[0381] Preferred is a recombinant vector comprising the chimeric genecomprising a promoter active in a plant operatively linked to aheterologous DNA molecule encoding a protoporphyrinogen oxidase (protox)selected from the group consisting of a wheat protox comprising thesequence set forth in SEQ ID NO:10, a soybean protox comprising thesequence set forth in SEQ ID NO:12, cotton protox comprising thesequence set forth in SEQ ID NO:16, a sugar beet protox comprising thesequence set forth in SEQ ID NO:18, an oilseed rape protox comprisingthe sequence set forth in SEQ ID NO:20, a rice protox comprising thesequence set forth in SEQ ID NO:22, a sorghum protox comprising thesequence set forth in SEQ ID NO:24, and a sugar cane protox comprisingthe sequence set forth in SEQ ID NO:37, wherein the vector is capable ofbeing stably transformed into a host cell.

[0382] Also preferred is recombinant vector comprising the chimeric genecomprising a promoter that is active in a plant operatively linked tothe DNA molecule encoding a modified protoporphyrinogen oxidase (protox)comprising a plant protox having a first amino acid substitution and asecond amino acid substitution; the first amino acid substitution havingthe property of conferring resistance to a protox inhibitor; and thesecond amino acid substitution having the property of enhancing theresistance conferred by the first amino acid substitution, wherein thevector is capable of being stably transformed into a plant cell.

[0383] Also encompassed by the present invention is a host cell stablytransformed with the vector according to the invention, wherein the hostcell is capable of expressing the DNA molecule. Preferred is a host cellselected from the group consisting of a plant cell, a bacterial cell, ayeast cell, and an insect cell.

[0384] The present invention is further directed to plants and theprogeny thereof, plant tissue and plant seeds tolerant to herbicidesthat inhibit the naturally occurring protox activity in these plants,wherein the tolerance is conferred by a gene expressing a modifiedinhibitor-resistant protox enzyme as taught herein. Representativeplants include any plants to which these herbicides may be applied fortheir normally intended purpose. Preferred are agronomically importantcrops, i.e., angiosperms and gymnosperms such as Arabidopsis, sugarcane, soybean, barley, cotton, tobacco, sugar beet, oilseed rape, maize,wheat, sorghum, rye, oats, tomato, potato, turf and forage grasses,millet, forage, and rice and the like. More preferred are agronomicallyimportant crops, i.e., angiosperms and gymnosperms such as Arabidopsis,cotton, soybean, oilseed rape, sugar beet, maize, rice, wheat, barley,oats, rye, sorghum, millet, turf, forage, turf grasses. Particularlypreferred are agronomically important crops, i.e., angiosperms andgymnosperms such as Arabidopsis, soybean, cotton, sugar beet, oilseedrape, maize, wheat, sorghum, and rice.

[0385] Preferred is a plant comprising the DNA molecule encoding amodified protoporphyrinogen oxidase (protox) comprising a plant protoxhaving a first amino acid substitution and a second amino acidsubstitution; the first amino acid substitution having the property ofconferring resistance to a protox inhibitor; and the second amino acidsubstitution having the property of enhancing the resistance conferredby the first amino acid substitution, wherein the DNA molecule isexpressed in the plant and confers upon the plant tolerance to aherbicide in amounts that inhibit naturally occurring protox activity.Preferred is a plant, wherein the DNA molecule replaces a correspondingnaturally occurring protox coding sequence. Comprised by the presentinvention is a plant and the progeny thereof comprising the chimericgene according to the invention, wherein the chimeric gene confers uponthe plant tolerance to a herbicide in amounts that inhibit naturallyoccurring protox activity.

[0386] Encompassed by the present invention are transgenic plant tissue,including plants and the progeny thereof, seeds, and cultured tissue,stably transformed with at least one chimeric gene according to theinvention. Preferred is transgenic plant tissue, including plants,seeds, and cultured tissue, stably transformed with at least onechimeric gene that comprises an expression cassette comprisingessentially a promoter, but especially a promoter that is active in aplant, operatively linked to the DNA molecule encoding anprotoporphyrinogen oxidase (protox) enzyme that is resistant toherbicides at levels that inhibit the corresponding unmodified versionof the enzyme in the plant tissue.

[0387] The present invention is further directed to plants, planttissue, plant seeds, and plant cells tolerant to herbicides that inhibitthe naturally occurring protox activity in these plants, wherein thetolerance is conferred by increasing expression of wild-typeherbicide-sensitive protox. This results in a level of a protox enzymein the plant cell at least sufficient to overcome growth inhibitioncaused by the herbicide. The level of expressed enzyme generally is atleast two times, preferably at least five times, and more preferably atleast ten times the natively expressed amount. Increased expression maybe due to multiple copies of a wild-type protox gene; multipleoccurrences of the coding sequence within the gene (i.e. geneamplification) or a mutation in the non-coding, regulatory sequence ofthe endogenous gene in the plant cell. Plants having such altered geneactivity can be obtained by direct selection in plants by methods knownin the art (see, e.g. U.S. Pat. No. 5,162,602, and U.S. Pat. No.4,761,373, and references cited therein). These plants also may beobtained by genetic engineering techniques known in the art. Increasedexpression of a herbicide-sensitive protox gene can also be accomplishedby stably transforming a plant cell with a recombinant or chimeric DNAmolecule comprising a promoter capable of driving expression of anassociated structural gene in a plant cell operatively linked to ahomologous or heterologous structural gene encoding the protox enzyme.

[0388] The recombinant DNA molecules of the invention can be introducedinto the plant cell in a number of art-recognized ways. Those skilled inthe art will appreciate that the choice of method might depend on thetype of plant, i.e. monocot or dicot, targeted for transformation.Suitable methods of transforming plant cells include microinjection(Crossway et al., BioTechniques 4:320-334 (1986)), electroporation(Riggs et al, Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986),Agrobacterium mediated transformation (Hinchee et al., Biotechnology6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J.3:2717-2722 (1984)), ballistic particle acceleration using devicesavailable from Agracetus, Inc., Madison, Wis. and Dupont, Inc.,Wilmington, Del. (see, for example, Sanford et al., U.S. Patent4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)),protoplast transformation/regeneration methods (see U.S. Pat. No.5,350,689 issued Sep. 27, 1994 to Ciba-Geigy Corp.), and pollentransformation (see U.S. Pat. No. 5,629,183). Also see, Weissinger etal., Annual Rev. Genet. 22:421-477 (1988); Sanford et al., ParticulateScience and Technology 5:27-37 (1987) (onion); Christou et al., PlantPhysiol. 87:671-674 (1988) (soybean); McCabe et al., Bio/Technology6:923-926 (1988) (soybean); Datta et al., Bio/Technology 8:736-740(1990) (rice); Klein et al., Proc. Natl. Acad. Sci. USA, 85:43054309(1988) (maize); Klein et al., Bio/Technology 6:559-563 (1988) (maize);Klein et al., Plant Physiol. 91:440-444 (1988) (maize); Fromm et al.,Bio/Technology 8:833-839 (1990); Gordon-Kamm et al., Plant Cell2:603-618 (1990) (maize); and U.S. Patent Nos. 5,591,616 and 5,679,558(rice).

[0389] Comprised within the scope of the present invention aretransgenic plants, in particular transgenic fertile plants transformedby means of the aforedescribed processes and their asexual and/or sexualprogeny, which still are resistant or at least tolerant to inhibition bya herbicide at levels that normally are inhibitory to the naturallyoccurring protox activity in the plant. Progeny plants also includeplants with a different genetic background than the parent plant, whichplants result from a backcrossing program and still comprise in theirgenome the herbicide resistance trait according to the invention. Veryespecially preferred are hybrid plants that are resistant or at leasttolerant to inhibition by a herbicide at levels that normally areinhibitory to the naturally occurring protox activity in the plant.

[0390] The transgenic plant according to the invention may be adicotyledonous or a monocotyledonous plant. Preferred aremonocotyledonous plants of the Graminaceae family involving Lolium, Zea,Triticum, Triticale, Sorghum, Saccharum, Bromus, Oryzae, Avena, Hordeum,Secale and Setaria plants. More preferred are transgenic maize, wheat,barley, sorghum, rye, oats, sugar cane, turf and forage grasses, milletand rice. Especially preferred are maize, wheat, sorghum, rye, oats,turf grasses and rice.

[0391] Among the dicotyledonous plants Arabidopsis, soybean, cotton,sugar beet, oilseed rape, tobacco, tomato, potato, and sunflower aremore preferred herein. Especially preferred are soybean, cotton,tobacco, sugar beet, tomato, potato, and oilseed rape.

[0392] The expression ‘progeny’ is understood to embrace both,“asexually” and “sexually” generated progeny of transgenic plants. Thisdefinition is also meant to include all mutants and variants obtainableby means of known processes, such as for example cell fusion or mutantselection and that still exhibit the characteristic properties of theinitial transformed plant, together with all crossing and fusionproducts of the transformed plant material. This also includes progenyplants that result from a backcrossing program, as long as the progenyplants still contain the herbicide resistant trait according to theinvention.

[0393] Another object of the invention concerns the proliferationmaterial of transgenic plants. The proliferation material of transgenicplants is defined relative to the invention as any plant material thatmay be propagated sexually or asexually in vivo or in vitro.Particularly preferred within the scope of the present invention areprotoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, eggcells, zygotes, together with any other propagating material obtainedfrom transgenic plants.

[0394] Parts of plants, such as for example flowers, stems, fruits,leaves, roots originating in transgenic plants or their progenypreviously transformed by means of the process of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

[0395] A further object of the invention is a method of producingplants, protoplasts, cells, calli, tissues, organs, seeds, embryos,pollen, egg cells, zygotes, together with any other propagatingmaterial, parts of plants, such as for example flowers, stems, fruits,leaves, roots originating in transgenic plants or their progenypreviously transformed by means of the process of the invention, whichtherefore produce an inhibitor resistant form of a plant protox enzymeby transforming the plant, plant parts with the DNA according to theinvention. Preferred is a method of producing a host cell comprising anisolated DNA molecule encoding a protein from a eukaryote havingprotoporphyrinogen oxidase (protox) activity comprising transforming thehost cell with a recombinant vector molecule according to the invention.Further preferred is a method of producing a plant cell comprising anisolated DNA molecule encoding a protein from a eukaryote havingprotoporphyrinogen oxidase (protox) activity comprising transforming theplant cell with a recombinant vector molecule according to theinvention. Preferred is a method of producing transgenic progeny of atransgenic parent plant comprising an isolated DNA molecule encoding aprotein from a eukaryote having protoporphyrinogen oxidase (protox)activity comprising transforming the parent plant with a recombinantvector molecule according to the invention and transferring theherbicide tolerant trait to the progeny of the transgenic parent plantinvolving known plant breeding techniques.

[0396] Preferred is a method for the production of plants, planttissues, plant seeds and plant parts, which produce aninhibitor-resistant form of the plant protox enzyme, wherein the plants,plant tissues, plant seeds and plant parts have been stably transformedwith a structural gene encoding the resistant protox enzyme.Particularly preferred is a method for the production of plants, planttissues, plant seeds and plant parts, wherein the plants, plant tissues,plant seeds and plant parts have been stably transformed with the DNAaccording to the invention. Especially preferred is a method for theproduction of the plants, plant tissues, plant seeds and plant parts,which produce an inhibitor-resistant form of the plant protox enzyme,wherein the plants, plant tissues, plant seeds and plant parts have beenprepared by direct selection techniques whereby herbicide resistantlines are isolated, characterized and developed.

[0397] The genetic properties engineered into the transgenic seeds andplants described above are passed on by sexual reproduction orvegetative growth and can thus be maintained and propagated in progenyplants. Generally the maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

[0398] Use of the advantageous genetic properties of the transgenicplants and seeds according to the invention can further be made in plantbreeding that aims at the development of plants with improved propertiessuch as tolerance of pests, herbicide tolerance, or stress tolerance,improved nutritional value, increased yield, or improved structurecausing less loss from lodging or shattering. The various breeding stepsare characterized by well-defined human intervention such as selectingthe lines to be crossed, directing pollination of the parental lines, orselecting appropriate progeny plants. Depending on the desiredproperties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines that for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow to dispense with the methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained that, due to their optimized genetic“equipment”, yield harvested product of better quality than productsthat were not able to tolerate comparable adverse developmentalconditions.

[0399] In seeds production germination quality and uniformity of seedsare essential product characteristics, whereas germination quality anduniformity of seeds harvested and sold by the farmer is not important.As it is difficult to keep a crop free from other crop and weed seeds,to control seedborne diseases, and to produce seed with goodgermination, fairly extensive and well-defined seed production practiceshave been developed by seed producers, who are experienced in the art ofgrowing, conditioning and marketing of pure seed. Thus, it is commonpractice for the farmer to buy certified seed meeting specific qualitystandards instead of using seed harvested from his own crop. Propagationmaterial to be used as seeds is customarily treated with a protectantcoating comprising herbicides, insecticides, fungicides, bactericides,nematicides, molluscicides or mixtures thereof. Customarily usedprotectant coatings comprise compounds such as captan, carboxin, thiram(TMTD®), methalaxyl (Apron®), and pirimiphos-methyl (Actellic®). Ifdesired these compounds are formulated together with further carriers,surfactants or application-promoting adjuvants customarily employed inthe art of formulation to provide protection against damage caused bybacterial, fungal or animal pests. The protectant coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

[0400] It is thus a further object of the present invention to provideplant propagation material for cultivated plants, but especially plantseed that is treated with an seed protectant coating customarily used inseed treatment.

[0401] It is a further aspect of the present invention to provide newagricultural methods such as the methods exemplified above, which arecharacterized by the use of transgenic plants, transgenic plantmaterial, or transgenic seed according to the present invention.Comprised by the present invention is an agricultural method, wherein atransgenic plant or the progeny thereof is used comprising a chimericgene according to the invention in an amount sufficient to expressherbicide resistant forms of herbicide target proteins in a plant toconfer tolerance to the herbicide.

[0402] To breed progeny from plants transformed according to the methodof the present invention, a method such as that which follows may beused: maize plants produced as described in the examples set forth beloware grown in pots in a greenhouse or in soil, as is known in the art,and permitted to flower. Pollen is obtained from the mature tassel andused to pollinate the ears of the same plant, sibling plants, or anydesirable maize plant. Similarly, the ear developing on the transformedplant may be pollinated by pollen obtained from the same plant, siblingplants, or any desirable maize plant. Transformed progeny obtained bythis method may be distinguished from non-transformed progeny by thepresence of the introduced gene(s) and/or accompanying DNA (genotype),or the phenotype conferred. The transformed progeny may similarly beselfed or crossed to other plants, as is normally done with any plantcarrying a desirable trait. Similarly, tobacco or other transformedplants produced by this method may be selfed or crossed as is known inthe art in order to produce progeny with desired characteristics.Similarly, other transgenic organisms produced by a combination of themethods known in the art and this invention may be bred as is known inthe art in order to produce progeny with desired characteristics.

[0403] The modified inhibitor-resistant protox enzymes of the inventionhave at least one amino acid substitution, addition or deletion relativeto their naturally occurring counterpart (i.e. inhibitor-sensitive formsthat occur naturally in a plant without being manipulated, eitherdirectly via recombinant DNA methodology or indirectly via selectivebreeding, etc., by man). Amino acid positions that may be modified toyield an inhibitor-resistant form of the protox enzyme, or enhanceinhibitor resistance, are indicated in bold type in Table 1A in thecontext of plant protox-1 sequences from Arabidopsis, maize, soybean,cotton, sugar beet, oilseed rape, rice, sorghum, wheat, and sugar cane.The skilled artisan will appreciate that equivalent changes may be madeto any plant protox gene having a structure sufficiently similar to theprotox enzyme sequences shown herein to allow alignment andidentification of those amino acids that are modified according to theinvention to generate inhibitor-resistant forms of the enzyme. Suchadditional plant protox genes may be obtained using standard techniquesas described in International application no. PCT/IB95/00452 filed Jun.8, 1995, published Dec. 21, 1995 as WO 95/34659 whose relevant parts areherein incorporated by reference.

[0404] DNA molecules encoding the herbicide resistant protox codingsequences taught herein may be genetically engineered for optimalexpression in a crop plant. This may include altering the codingsequence of the resistance allele for optimal expression in the cropspecies of interest. Methods for modifying coding sequences to achieveoptimal expression in a particular crop species are well known (see,e.g. Perlak et al., Proc. Natl. Acad. Sci. USA 88: 3324 (199 1); Kozielet al., Bio/technol. 11: 194 (1993)).

[0405] Genetically engineering a protox coding sequence for optimalexpression may also include operatively linking the appropriateregulatory sequences (i.e. promoter, signal sequence, transcriptionalterminators). Examples of promoters capable of functioning in plants orplant cells (i.e., those capable of driving expression of the associatedstructural genes such as protox in plant cells) include the cauliflowermosaic virus (CAMV) 19S or 35S promoters and CAMV double promoters;nopaline synthase promoters; pathogenesis-related (PR) proteinpromoters; small subunit of ribulose bisphosphate carboxylase(ssuRUBISCO) promoters, heat shock protein promoter from Brassica withreference to EPA 0 559 603 (hsp8o promoter), Arabidopsis actin promoterand the SuperMas promoter with reference to WO 95/14098 and the like.Preferred promoters will be those that confer high level constitutiveexpression or, more preferably, those that confer specific high levelexpression in the tissues susceptible to damage by the herbicide.Preferred promoters are the rice actin promoter (McElroy et al., Mol.Gen. Genet. 231: 150 (1991)), maize ubiquitin promoter (EP 0 342 926;Taylor et al., Plant Cell Rep. 12: 491 (1993)), and the PR-1 promoterfrom tobacco, Arabidopsis, or maize (see U.S. Pat. No. 5,614,395 toRyals et al., incorporated by reference herein in its entirety). Thepromoters themselves may be modified to manipulate promoter strength toincrease protox expression, in accordance with art-recognizedprocedures.

[0406] The inventors have also discovered that another preferredpromoter for use with the inhibitor-resistant protox coding sequences isthe promoter associated with the native protox gene (i.e. the protoxpromoter; see copending, co-owned U.S. patent application Ser. No.08/808,323, entitled “Promoters from Protoporphyrinogen Oxidase Genes”,incorporated by reference herein in its entirety.) The promoter sequencefrom an Arabidopsis protox-1 gene is set forth in SEQ ID NO:13, thepromoter sequence from a maize protox-1 gene is set forth in SEQ IDNO:14, and the promoter sequence from a sugar beet protox-1 gene is setforth in SEQ ID NO:26.

[0407] Since the protox promoter itself is suitable for expression ofinhibitor-resistant protox coding sequences, the modifications taughtherein may be made directly on the native protox gene present in theplant cell genome without the need to construct a chimeric gene withheterologous regulatory sequences. Such modifications can be made viadirected mutagenesis techniques such as homologous recombination andselected for based on the resulting herbicide-resistance phenotype (see,e.g. Example 10, Pazkowski et al., EMBO J. 7. 4021-4026 (1988), and U.S.Pat. No. 5,487,992, particularly columns 18-19 and Example 8). An addedadvantage of this approach is that besides containing the native protoxpromoter, the resulting modified gene will also include any otherregulatory elements, such as signal or transit peptide coding sequences,which are part of the native gene.

[0408] In the event of transformation of the nuclear genome, signal ortransit peptides may be fused to the protox coding sequence in chimericDNA constructs of the invention to direct transport of the expressedprotox enzyme to the desired site of action. Examples of signal peptidesinclude those natively linked to the plant pathogenesis-relatedproteins, e.g. PR-1, PR-2, and the like. See, e.g., Payne et al., PlantMol. Biol. 11:89-94 (1988). Examples of transit peptides include thechloroplast transit peptides such as those described in Von Heijne etal., Plant Mol. Biol. Rep. 9:104-126 (1991); Mazur et al., PlantPhysiol. 85: 1110 (1987); Vorst et al., Gene 65: 59 (1988), andmitochondrial transit peptides such as those described in Boutry et al.,Nature 328:340-342 (1987). Chloroplast and mitochondrial transitpeptides are contemplated to be particularly useful with the presentinvention as protox enzymatic activity typically occurs within themitochondria and chloroplast. Most preferred for use are chloroplasttransit peptides, as inhibition of the protox enzymatic activity in thechloroplasts is contemplated to be the primary basis for the action ofprotox-inhibiting herbicides (Witkowski and Halling, Plant Physiol. 87:632 (1988); Lehnen et al., Pestic. Biochem. Physiol. 37: 239 (1990);Duke et al., Weed Sci. 39: 465 (1991)). Also included are sequences thatresult in localization of the encoded protein to various cellularcompartments such as the vacuole. See, for example, Neuhaus et al.,Proc. Natl. Acad. Sci. USA 88: 10362-10366 (1991) and Chrispeels, Ann.Rev. Plant Physiol. Plant Mol. Biol. 42: 21-53 (1991). The relevantdisclosures of these publications are incorporated herein by referencein their entirety.

[0409] Chimeric genes of the invention may contain multiple copies of apromoter or multiple copies of the protox structural genes. In addition,the construct(s) may include coding sequences for markers and codingsequences for other peptides such as signal or transit peptides, each inproper reading frame with the other functional elements in the DNAmolecule. The preparation of such constructs are within the ordinarylevel of skill in the art.

[0410] Useful markers include peptides providing herbicide, antibioticor drug resistance, such as, for example, resistance to hygromycin,kanamycin, G418, gentamycin, lincomycin, methotrexate, glyphosate,phosphinothricin, or the like. These markers can be used to select cellstransformed with the chimeric DNA constructs of the invention fromuntransformed cells. Other useful markers are peptidic enzymes that canbe easily detected by a visible reaction, for example a color reaction,for example luciferase, β-glucuronidase, or β-galactosidase.

[0411] The method of positive selection of genetically transformed cellsinto which a desired nucleotide sequence can be incorporated byproviding the transformed cells with a selective advantage is hereinincorporated by reference as WO 94/20627.

[0412] Where a herbicide resistant protox allele is obtained viadirected mutation of the native gene in a crop plant or plant cellculture from which a crop plant can be regenerated, it may be moved intocommercial varieties using traditional breeding techniques to develop aherbicide tolerant crop without the need for genetically engineering themodified coding sequence and transforming it into the plant.Alternatively, the herbicide resistant gene may be isolated, geneticallyengineered for optimal expression and then transformed into the desiredvariety.

[0413] Genes encoding altered protox resistant to a protox inhibitor canalso be used as selectable markers in plant cell transformation methods.For example, plants, plant tissue or plant cells transformed with atransgene can also be transformed with a gene encoding an altered protoxcapable of being expressed by the plant. The thus-transformed cells aretransferred to medium containing the protox inhibitor wherein only thetransformed cells will survive. Protox inhibitors contemplated to beparticularly useful as selective agents are the diphenylethers {e.g.acifluorfen, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid;its methyl ester; or oxyfluorfen,2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles,(e.g. oxidiazon, 3-[2,4-dichloro-5-(1 -methylethoxy)phenyl]-5-( 1,1-dimethylethyl)-1,3,4 -oxadiazol-2-(3H)-one), cyclic imides (e.g.S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and its0-phenylpyrrolidino- and piperidinocarbamate analogs and bicyclictriazolones as disclosed in the International patent application WO92/04827; EP 532146).

[0414] The method is applicable to any plant cell capable of beingtransformed with an altered protox-encoding gene, and can be used withany transgene of interest. Expression of the transgene and the protoxgene can be driven by the same promoter functional on plant cells, or byseparate promoters.

[0415] Modified inhibitor-resistant protox enzymes of the presentinvention are resistant to herbicides that inhibit the naturallyoccurring protox activity. The herbicides that inhibit protox includemany different structural classes of molecules (Duke et al., Weed Sci.39: 465 (1991); Nandihalli et al., Pesticide Biochem. Physiol. 43: 193(1992); Matringe et al., FEBS Lett. 245: 35 (1989); Yanase and Andoh,Pesticide Biochem. Physiol. 35: 70 (1989)), including the diphenylethers{e.g. acifluorifen,5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobezoic acid; its methylester; or oxyfluorfen,2-chloro-1-(3-ethoxy4-nitrophenoxy)-4-(trifluorobenzene) }, oxidiazoles(e.g. oxidiazon,3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one),cyclic imides (e.g. S-23142,N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide;chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide),phenyl pyrazoles (e.g. TNPP-ethyl, ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate; M&B39279), pyridine derivatives (e.g. LS 82-556), and phenopylate and itsO-phenylpyrrolidino- and piperidinocarbamate analogs.

[0416] The diphenylethers of particular significance are those havingthe general formula

[0417] wherein R equals —COONa (Formula II), —CONHSO₂CH₃ (Formula III)or —COOCH₂COOC₂H₅ (Formula IV; see Maigrot et al., Brighton CropProtection Conference-Weeds: 47-51 (1989)).

[0418] Additional diphenylethers of interest are those where R equals:

[0419] (Formula IVa; see Hayashi et al., Brighton Crop ProtectionConference-Weeds: 53-58 (1989)).

[0420] An additional diphenylether of interest is one having theformula:

[0421] (Formula IVb; bifenox, see Dest et al., Proc. Northeast Weed Sci.Conf 27: 31 (1973)).

[0422] A further diphenylether of interest is one having the formula:

[0423] (Formula IVc; oxyfluorfen; see Yih and Swithenbank, J. Agric.Food Chem., 23: 592 (1975))

[0424] Yet another diphenylether of interest is one having the formula:

[0425] (Formula IVd; lactofen, see page 623 of “The Pesticide Manual”,10^(th) ed., ed. by C. Tomlin, British Crop Protection Council, Surrey(1994))

[0426] Also of significance are the class of herbicides known as imides,having the general formula

[0427] wherein Q equals

[0428] (see Hemper et al. (1995) in “Proceedings of the EighthInternational Congress of Pesticide Chemistry”, Ragdale et al., eds.,Amer. Chem. Soc, Washington, D.C., pp.42-48 (1994)); and R₁ equals H, Clor F, R₂ equals Cl and R₃ is an optimally substituted ether, thioether,ester, amino or alkyl group. Alternatively, R₂ and R₃ together may forma 5 or 6 membered heterocyclic ring. Examples of imide herbicides ofparticular interest are

[0429] (Formula VIIa; fluthiacet-methyl, see Miyazawa et al., BrightonCrop Protection Conference-Weeds, pp. 23-28 (1993))

[0430] Crop Protection Conference-Weeds, pp. 77-82 (1991)).

[0431] The herbicidal activity of the above compounds is described inthe Proceedings of the 1991 Brighton Crop Protection Conference, Weeds(British Crop Protection Council) (Formulae X and XVI), Proceedings ofthe 1993 Brighton Crop Protection Conference, Weeds (British CropProtection Council) (Formulae XII and XIII), U.S. Pat. No. 4,746,352(Formula XI) and Abstracts of the Weed Science Society of America vol.33, pg. 9 (1993) (Formula XIV).

[0432] The most preferred imide herbicides are those classified asaryluracils and having the general formula

[0433] wherein R signifies the group(C₂₋₆-alkenyloxy)carbonyl-C₁₋₄-alkyl, as disclosed in U.S. Pat. No.5,183,492, herein incorporated by reference.

[0434] Also of significance are herbicides having the general formula:

[0435] wherein

[0436] R₁ is C₁-C₄-alkyl, optionally substituted by one or more halogenatoms;

[0437] R₂ is hydrogen, or a C₁-C₄-alkoxy, each of which is optionallysubstituted by one or more halogen atoms, or

[0438] R₁ and R₂ together from the group —(CH₂)_(n)—X—, where X is boundat R₂;

[0439] R₃ is hydrogen or halogen,

[0440] R₄ is hydrogen or C₁-C₄-alkyl,

[0441] R₅ is hydrogen, nitro, cyano or the group —COOR₆ or —CONR₇R₈, and

[0442] R₆ is hydrogen, C₁-C₆-alkyl, C₂-C₆-alkenyl or C₂-C₆-alkynyl;

[0443] (see international patent publications WO 94/08999, WO 93/10100,and U.S. Pat. No. 5,405,829 assigned to Schering);

[0444] and 3-substituted-2-aryl-4,5,6,7-tetrahydroindazoles (Lyga et al.Pesticide Sci. 42:29-36 (1994)).

[0445] Also of significance are phenylpyrazoles of the type described inWO 96/01254 and WO 97/00246, both of which are hereby incorporated byreference. (Formula XXII).

[0446] Also of significance are pyridyl pyrazoles such as the following:

[0447] Levels of herbicide that normally are inhibitory to the activityof protox include application rates known in the art, and that dependpartly on external factors such as environment, time and method ofapplication. For example, in the case of the imide herbicidesrepresented by Formulae V through IX, and more particularly thoserepresented by Formulae X through XVII, the application rates range from0.0001 to 10 kg/ha, preferably from 0.005 to 2 kg/ha. This dosage rateor concentration of herbicide may be different, depending on the desiredaction and particular compound used, and can be determined by methodsknown in the art.

[0448] A further object of the invention is a method for controlling thegrowth of undesired vegetation that comprises applying to a populationof the plant selected from a group consisting of Arabidopsis, sugarcane, soybean, barley, cotton, tobacco, sugar beet, oilseed rape, maize,wheat, sorghum, rye, oats, turf and forage grasses , millet, forage andrice and the like an effective amount of a protox-inhibiting herbicide.Preferred is a method for controlling the growth of undesiredvegetation, which comprises applying to a population of the selectedfrom the group consisting of selected from the group consisting ofsoybean, cotton, tobacco, sugar beet, oilseed rape, maize, wheat,sorghum, rye, oats, turf grasses and rice an effective amount of aprotox-inhibiting herbicide. Particularly preferred is a method forcontrolling the growth of undesired vegetation, which comprises applyingto a population of the selected from the group consisting ofArabidopsis, soybean, cotton, sugar beet, oilseed rape, maize, wheat,sorghum, and rice.

[0449] III. Plastid Transformation and Expression

[0450] The present invention further encompasses a chimeric genecomprising a promoter capable of expression in a plant plastidoperatively linked to a DNA molecule of the present invention. Apreferred promoter capable of expression in a plant plastid is apromoter isolated from the 5′ flanking region upstream of the codingregion of a plastid gene, which may come from the same or a differentspecies, and the native product of which is typically found in amajority of plastid types including those present in non-green tissues.Examples of such promoters are promoters of cLpP genes, such as thetobacco clpP gene promoter (WO 97106250, incorporated herein byreference) and the Arabidopsis clpP gene promoter (U.S. application Ser.No. 09/038,878, incorporated herein by reference). Other promoters thatare capable of expressing a DNA molecule in plant plastids are promotersrecognized by viral RNA polymerases. Preferred promoters of this typeare promoters recognized by a single sub-unit RNA polymerase, such asthe T7 gene 10 promoter, which is recognized by the bacteriophage T7DNA-dependent RNA polymerase. Yet another promoter that is capable ofexpressing a DNA molecule in plant plastids comes from the regulatoryregion of the plastid 16S ribosomal RNA operon (Harris et al.,Microbiol. Rev. 58:700-754 (1994), Shinozaki et al., EMBO J. 5:2043-2049(1986), both of which are incorporated herein by reference). The geneencoding the T7 polymerase is preferably transformed into the nucleargenome and the T7 polymerase is targeted to the plastids using a plastidtransit peptide. Expression of the DNA molecules in the plastids can beconstitutive or can be inducible. These different embodiment areextensively described in WO 98/11235, incorporated herein by reference.The chimeric gene preferably further comprises a 5′ untranslatedsequence (5′ UTR) functional in plant plastids and a plastid gene 3′untranslated sequence (3′ UTR) operatively linked to a DNA molecule ofthe present invention. Preferably, the 3′ UTR is a plastid rps16 gene 3′untranslated sequence. In a further embodiment, the chimeric genecomprises a poly-G tract instead of a 3′ untranslated sequence.

[0451] The present invention also encompasses a plastid transformationvector comprising the chimeric gene described above and flanking regionsfor integration into the plastid genome by homologous recombination. Theplastid transformation vector may optionally comprise at least onechloroplast origin of replication. The present invention alsoencompasses a plant plastid transformed with such a plastidtransformation vector, wherein the DNA molecule is expressible in theplant plastid. The invention also encompasses a plant or plant cell,including the progeny thereof, comprising this plant plastid. In apreferred embodiment, the plant is homoplasmic for transgenic plastids.The plants transformed in the present invention may be monocots ordicots.

[0452] A preferred monocot is maize and a preferred dicot is tobacco.Other preferred dicots are tomato and potato.

[0453] In a preferred embodiment, the present invention encompasses achimeric gene comprising a promoter capable of expression in a plantplastid operatively linked to a DNA molecule isolated from a prokaryoteor a eukaryote that encodes a native or modified protox enzyme, such asa DNA molecule that encodes a native or modified wheat, soybean, cotton,sugar beet, oilseed rape, rice, sorghum, or sugar cane protox enzyme.Such a DNA molecule is comprised in a plastid transformation vector asdescribed above and plants homoplasmic for transgenic plastid genomesare produced. Expression in plant plastids of a DNA molecule thatencodes a modified protox enzyme preferably confers upon the planttolerance to a herbicide in amounts that inhibit naturally occurringprotox activity.

[0454] In a further preferred embodiment, the present inventionencompasses a chimeric gene comprising (a) a DNA molecule isolated froma plant, which in its native state encodes a polypeptide that comprisesa plastid transit peptide, and a mature enzyme that is natively targetedto a plastid of the plant by the plastid transit peptide, wherein theDNA molecule is modified such that it does not encode a functionalplastid transit peptide; and (b) a promoter capable of expressing theDNA molecule in a plastid, wherein the promoter is operatively linked tothe DNA molecule. In one preferred embodiment, the transit peptide ismutated and thus does not allow the proper transport of the enzymeencoded by the DNA molecule to the desired cell compartment, such as theplastid. In another preferred embodiment, a portion of the transitpeptide coding sequence or the entire transit peptide coding sequence isremoved from the DNA molecule, preventing the enzyme from being properlytargeted to the desired cell compartment.

[0455] The chimeric genes described above are inserted in plastidtransformation vectors, and the present invention is therefore alsodirected to plants having their plastid genome transformed with suchvectors, whereby the DNA molecule is expressible in plant plastids. Suchplants are preferably homoplasmic for transgenic plastids.

[0456] In a preferred embodiment, a DNA molecule described immediatelyabove encodes an enzyme that in its wild-type form is inhibited by aherbicide. In a further preferred embodiment, the DNA molecule encodesan enzyme that in its wild-type form is inhibited by a herbicide, butthat comprises at least one amino acid change compared to the wild-typeenzyme. Such an amino acid change makes the enzyme resistant tocompounds that naturally inhibit the wild-type enzyme. In a furtherpreferred embodiment, the DNA molecule encodes an enzyme havingprotoporphyrinogen oxidase (protox) activity. In a further preferredembodiment, the transit peptide is removed from the DNA molecule asfurther illustrated in Examples 37-42. Plants homoplasmic for transgenicplastids of the invention are resistant to high amounts of herbicidessuch as Formula XVII that inhibit the naturally occurring protoxactivity (as further illustrated in Example 44).

[0457] In another preferred embodiment, the transit peptide of a DNAmolecule encoding a 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPsynthase) is mutated or removed. The resulting DNA molecule is fused toa promoter capable of expression in plant plastids and homoplasmicplants harboring such constructs in their plastid genomes are obtained.These plants are resistant to herbicidal compounds that naturallyinhibit EPSP synthase, in particular glyphosate. In another preferredembodiment, the transit peptide of a DNA molecule encoding aacetolactate synthase (ALS) is mutated or removed. The resulting DNAmolecule is fused to a promoter capable of expression in plant plastidsand homoplasmic plants harboring such constructs in their plastid genomeare obtained. These plants are resistant to herbicidal compounds thatnaturally inhibit ALS, in particular sulfonylureas. In another preferredembodiment, the transit peptide of a DNA molecule encoding aacetoxyhydroxyacid synthase (AHAS) is mutated or removed. The resultingDNA molecule is fused to a promoter capable of expression in plantplastids and homoplasmic plants harboring such constructs in theirplastid genome are obtained. These plants are resistant to herbicidalcompounds that naturally inhibit AHAS, in particular, imidazolinone andsulfonamide herbicides. In another preferred embodiment, the transitpeptide of a DNA molecule encoding an acetylcoenzyme A carboxylase(ACCase) is mutated or removed. The resulting DNA molecule is fused to apromoter capable of expression in plant plastids and homoplasmic plantsharboring such constructs in their plastid genome are obtained. Theseplants are resistant to herbicidal compounds that naturally inhibitACCase, in particular cyclohexanedione and arylphenoxypropanoic acidherbicides. In another preferred embodiment, the transit peptide of aDNA molecule encoding a glutamine synthase (GS) is mutated or removed.The resulting DNA molecule is fused to a promoter capable of expressionin plant plastids and homoplasmic plants harboring such constructs intheir plastid genome are obtained. These plants are resistant toherbicidal compounds that naturally inhibit GS, in particularphosphinothricin and methionine sulfoximine.

[0458] The present invention is also further directed to a method ofobtaining herbicide-resistant plants by transforming their plastidgenome with a chimeric gene comprising (a) a DNA molecule isolated froma plant, which in its native state encodes a polypeptide that comprisesa plastid transit peptide, and a mature enzyme that is natively targetedto a plastid of the plant by the plastid transit peptide, wherein theDNA molecule is modified such that it does not encode a functionalplastid transit peptide; and (b) a promoter capable of expressing theDNA molecule in a plastid, wherein the promoter is operatively linked tothe DNA molecule. Examples of enzymes that are used in the presentinvention are cited immediately above, but the applicability of such amethod is not limited to the cited examples.

[0459] The present invention is still further directed to a novel methodfor selecting a transplastomic plant cell, comprising the steps of:introducing the above-described chimeric gene into the plastome of aplant cell; expressing the encoded enzyme in the plastids of the plantcell; and selecting a cell that is resistant to a herbicidal compoundthat naturally inhibits the activity of the enzyme, whereby theresistant cell comprises transformed plastids. In a preferredembodiment, the enzyme is naturally inhibited by a herbicidal compoundand the transgenic plant is able to grow on an amount of the herbicidalcompound that naturally inhibits the activity of the enzyme. In afurther preferred embodiment, the enzyme has protoporphyrinogen oxidase(protox) activity and is modified so that it that confers resistance toprotox inhibitors.

[0460] A further aspect of the present invention is a novel method forplastid transformation of recalcitrant plants. The methods pioneered forplastid transformation of tobacco and lower plant species rely onnon-lethal selection for resistance to antibiotics that preferentiallyaffect the plastid translational apparatus and hence allowphoto-heterotrophic transformants to outgrow heterotrophic,non-transformed tissue.

[0461] Several factors have likely contributed to the difficultiesencountered with plastid transformation of monocots and other dicots.For example, the maize chloroplast 16S ribosomal RNA (rRNA) is naturallyresistant to spectinomycin because of the presence of a G at position1138 in the Zea mays 16S rDNA gene (Harris et al., 1994). Thus,utilization of 16s rRNA point mutations that confer spectinomycin and/orstreptomycin resistance which have been used successfully as selectablechloroplast markers in Chlamydomonas and tobacco (Boynton and Gillham(1993) In Wu, R. [Ed.]Methods in Enzymology Vol 217. Academic Press, SanDiego, pp. 510-536; Svab et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8526-8530) is not feasible for maize. Natural spectinomycin andstreptomycin resistance in maize also obviates the use of the bacterialaada gene encoding aminoglycoside 3′-adenyltransferase, which results indominant spectinomycin and streptomycin resistance and allows a 100-foldincrease in tobacco chloroplast transformation efficiency (Svab andMaliga (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 913-917). Use ofkanamycin (the only other antibiotic proven to be useful for chloroplasttransformation) is also problematic due to a large excess (ca. 50: 1) ofnuclear vs. chloroplast-encoded resistance in tobacco followingbombardment of the bacterial nptII gene encoding neomycinphosphotransferase (Carrer et al. (1993) Mol. Gen. Genet. 241: 49-56).This has been shown to result from both a high frequency of spontaneousnuclear resistance mutants as well as integration of nptli into thenuclear genome. Since nptII is also a highly effective selectable markerfor maize nuclear transformation it is reasonable to expect similarbackground levels to that observed in tobacco. Spontaneous resistanceand a significant excess of selectable marker integration by random,illegitimate recombination into the nuclear genome, rather thanhomologous integration into the chloroplast genome, would make recoveryof bona fide chloroplast transformants difficult if not impossible.

[0462] A more fundamental reason for the difficulties encountered withplastid transformation in plant species other than tobacco may have todo with the non-photosynthetic nature of many regenerable cultured planttissues, especially in maize and Arabidopsis. Tobacco is an exception inthat cultured vegetative tissues are regenerable and contain maturedifferentiated chloroplasts that are photosynthetically competent in thepresence of sucrose. Consequently, the current system for selectingtobacco plastid transformants relies on the faster growth rate oftransformed cells that can use both reduced and inorganic carbonsources. Moreover, transformed cells do not suffer the chloroplastmembrane damage that results from inhibition of plastid proteinsynthesis in the light. This expression of selectable markers that actpreferentially on photosynthetic cells, driven by promoters that havehigh activity in differentiated chloroplasts, is unlikely to work innon-green tissues containing proplastids (e.g. dark-grown maize Type Icallus, somatic embryos) or amyloplasts/leucoplasts (e.g. Arabidopsisroot cultures). Plastid transformation in these plants requires aselectable marker that gives strong selection in all plastid types.

[0463] A preferred selectable marker for generalized plastidtransformation: (1) is active only in the plastid to eliminatenuclear-transformed “escapes”; (2) has a mode of action that does notdepend on photosynthetic competence or the presence of fullydifferentiated chloroplasts; and (3) has a level of resistance that isco-dependent on an adjustable external parameter (e.g. light), ratherthan being determined solely by the bulk concentration of a selectiveagent, so that selection pressure can vary during selection tofacilitate segregation of the many-thousand plastid genome copies.

[0464] In a preferred embodiment, such a selectable marker gene involvesthe use of a chimeric gene comprising an isolated DNA molecule encodinga plastid-targeted enzyme having in its natural state a plastid transitpeptide, wherein the DNA molecule is modified such that the transitpeptide either is absent or does not function to target the enzyme tothe plastid, wherein the DNA molecule is operatively linked to apromoter capable of expression in plant plastids. In a preferredembodiment, a DNA molecule of the present invention encodes an enzymethat is naturally inhibited by a herbicide. In another preferredembodiment, the DNA molecule encodes a protoporphyrinogen IX oxidase(“protox”). In a preferred embodiment, the protoporphyrinogen IX oxidasegene is from Arabidopsis thaliana and in a more preferred embodiment,the protoporphyrinogen IX oxidase gene is from Arabidopsis thaliana andcomprises at least one amino acid substitution. Preferably, an aminoacid substitution results in tolerance of the enzyme against inhibitionby an herbicide which naturally inhibits the activity of the enzyme. Lowconcentrations of herbicide are thought to kill wildtype plants due tolight-sensitive intermediates which build up when the plastid4ocalizedprotox enzyme is inhibited. Production of these photosensitizingcompounds does not require differentiated chloroplasts or activephotosynthesis, which is a key factor for successful plastidtransformation of plants whose regenerable cultured tissues are ofnon-photosynthetic nature.

[0465] Another key feature is to have expression of the selectablemarker gene in non-green plastids. In a preferred embodiment, theinvention encompasses the use of promoters that are capable ofexpression of operatively linked DNA molecules in plastids of both greenand non-green tissue. In particular, one such promoter comes from theregulatory region of the plastid 16S ribosomal RNA operon. Anothercandidate is the promoter and 5′ UTR from the plastid clpP gene. TheclpP gene product is expressed constitutively in plastids from all planttissues, including those that do not contain chloroplasts (Shanklin(1995) Plant Cell 7: 1713-22).

[0466] Other DNA molecules may be co-introduced in plant plastids usingthe method described above. In a preferred embodiment, a plastidtransformation vector of the present invention contains a chimeric geneallowing for selection of transformants as described above and at leastone other gene fused to a promoter capable of expression in plantplastids. The other such gene may, for example, confer resistance toinsect pests, or to fungal or bacterial pathogens, or may encode one ormore value-added traits.

EXAMPLES

[0467] The invention will be further described by reference to thefollowing detailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Ausubel (ed.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc.(1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor,N.Y. (1989); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984).

Section A. Isolation And Characterization Of Plant ProtoporphyrinogenOxidase (Protox) Genes Example 1 Isolation of a Wheat Protox-1 cDNABased on Sequence Homology to a Maize Protox-1 Coding Sequence

[0468] Total RNA prepared from Triticum aestivum (cv Kanzler) wassubmitted to Clontech for custom cDNA library construction in the LambdaUni-Zap vector. Approximately 50,000 pfu of the cDNA library were platedat a density of approximately 5,000 pfu per 10 cm Petri dish andduplicate filter lifts were made onto nitrocellulose membranes(Schleicher and Schuell). The plaque lifts were probed with the maizeprotox-1 cDNA (SEQ ID NO:5; see Example 2 of International applicationno. PCT/IB95/00452, filed Jun. 8, 1995, published Dec. 21, 1995 as WO95/34659) labeled with 32P-dCTP by the random priming method (LifeTechnologies). Hybridization conditions were 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C. Wash conditions were2×SSC, 1% SDS at 50° C. (Church and Gilbert, Proc. Natl. Acad. Sci. USA81: 1991-1995 (1984), hereby incorporated by reference in its entirety.)Positively hybridizing plaques were purified and in vivo excised intopBluescript plasmids. The sequences of the cDNA inserts were determinedby the chain termination method using dideoxy terminators labeled withfluorescent dyes (Applied Biosystems, Inc.). The longest wheat protox-1cDNA obtained from initial screening efforts, designated “wheatprotox-1”, was 1489-bp in length. Wheat protox-1 lacks coding sequencefor the transit peptide plus approximately 126 amino acids of the maturecoding sequence based on comparison with the other known plant protoxpeptide sequences.

[0469] A second screen was performed to obtain a longer wheat protoxcDNA. For this screen, a Triticum aestivum (cv Kanzler) cDNA library wasprepared internally using the lambda Uni-Zap vector. Approximately200,000 pfu of the cDNA library was screened as indicated above, exceptthat the wheat protox-1 cDNA was used as a probe and hybridization andwash conditions were at 65° C. instead of 50° C. The longest wheat cDNAobtained from this screening effort, designated “wheat protox-1 a”, was1811-bp in length. The nucleotide sequence of this cDNA and the aminoacid sequence it encodes are set forth in SEQ ID NOs:9 and 10,respectively. Based on comparison with the other known plant protoxpeptide sequences and with corresponding genomic sequence, this cDNA iseither full-length or missing only a few transit peptide codons (Table1A). This wheat protein sequence is 91% identical (95% similar) to themaize protox-1 protein sequence set forth in SEQ ID NO:6.

[0470] Wheat protox-la, in the pBluescript SK vector, was deposited Mar.19, 1996, as pWDC-13 (NRRL #B21545).

Example 2 Isolation of a Soybean Protox-1 cDNA Based on SequenceHomology to an Arabidopsis Protox-1 Coding Sequence

[0471] A Lambda Uni-Zap cDNA library prepared from soybean (v Williams82, epicotyls) was purchased from Stratagene. Approximately 50,000 pfuof the library was plated at a density of approximately 5,000 pfu per 10cm Petri dish and duplicate filter lifts were made onto Colony/PlaqueScreen membranes (NEN Dupont). The plaque lifts were probed with theArabidopsis protox-1 cDNA (SEQ ID NO:1; see Example 1 of Internationalapplication no. PCT/IB95/00452, filed Jun. 8, 1995, published Dec. 21,1995 as WO 95/34659)) labeled with 32P-dCTP by the random priming method(Life Technologies). Hybridization conditions were 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C. Wash conditionswere 2×SSC, 1% SDS at 50° C. (Church and Gilbert (1984)). Positivelyhybridizing plaques were purified and in vivo excised into pBluescriptplasmids. The sequence of the cDNA inserts was determined by the chaintermination method using dideoxy terminators labeled with fluorescentdyes (Applied Biosystems, Inc.). The longest soybean cDNA obtained,designated “soybean protox-1”, is full-length based on comparison withthe other known plant protox peptide sequences (Table 1A). Soybeanprotox-1 is 1847-bp in length and encodes a protein of 58.8 kDa. Thenucleotide sequence of this cDNA and the amino acid sequence it encodesare set forth in SEQ ID NOs:11 and 12, respectively. The soybean proteinis 78% identical (87% similar) to the Arabidopsis protox-1 protein.

[0472] Soybean protox-1, in the pBluescript SK vector, was depositedDecember 15, 1995 as pWDC-12 (NRRL#B-21516).

Example 3 Isolation of a Cotton Protox-1 cDNA Based on Sequence Homologyto a Maize Protox-1 Coding Sequence

[0473] A Lambda Uni-Zap cDNA library prepared from Gossypium hirsutum L.(72 hr. dark grown cotyledons) was obtained from Dr. Dick Trelease,Dept. of Botany, Arizona State University (Ni W. and Trelease R. N.,Arch. Biochem. Biophys. 289: 237-243 (1991)). Approximately 50,000 pfuof the library was plated at a density of approximately 5,000 pfu per 10cm Petri dish and duplicate filter lifts were made onto Colony/PlaqueScreen membranes (NEN Dupont). The plaque lifts were probed with themaize protox-1 cDNA (SEQ ID NO:5) labeled with 32P-dCTP by the randompriming method (Life Technologies). Hybridization conditions were 7%sodium dodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C.Wash conditions were 2×SSC, 1% SDS at 500 C. (Church and Gilbert(1984)). Positively hybridizing plaques were purified and in vivoexcised into pBluescript plasmids. The sequence of the cDNA inserts wasdetermined by the chain termination method using dideoxy terminatorslabeled with fluorescent dyes (Applied Biosystems, Inc.). The longestcotton cDNA obtained, designated “cotton protox-1”, appears to befull-length based on comparison with the other known plant protoxpeptide sequences (Table 1A). Cotton protox-1 is 1826-bp in length andencodes a protein of 58.2 kDa. The nucleotide sequence of this cDNA andthe amino acid sequence it encodes are set forth in SEQ ID NOs:13 and14, respectively. The cotton protein is 77% identical (86% similar) tothe maize protox-1 protein.

[0474] Cotton protox-1, in the pBluescript SK vector, was deposited Jul.1, 1996 as pWDC-15 (NRRL #B-21594).

Example 4 Isolation of a Sugar Beet Protox-1 cDNA Based on SequenceHomology to an Arabidopsis Protox-1 Coding Sequence

[0475] A Lambda-Zap cDNA library prepared from Beta vulgaris wasobtained from Dr. Philip Rea, Dept. of Botany, Plant Science Institute,Philadelphia, Pa. (Yongcheol Kim, Eugene J. Kim, and Philip A. Rea,Plant Physiol. 106: 375-382 (1994)). Approximately 50,000 pfu of thecDNA library were plated at a density of approximately 5,000 pfu per 10cm Petri dish and duplicate filter lifts were made onto nitrocellulosemembranes (Schleicher and Schuell). The plaque lifts were probed withthe Arabidopsis protox-1 cDNA (SEQ ID NO:1) labeled with 32P-dCTP by therandom priming method (Life Technologies). Hybridization conditions were7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C.Wash conditions were 2×SSC, 1% SDS at 50° C. (Church and Gilbert(1984)). Positively hybridizing plaques were purified and in vivoexcised into pBluescript plasmids. The sequences of the cDNA insertswere determined by the chain termination method using dideoxyterminators labeled with fluorescent dyes (Applied Biosystems, Inc.).The longest sugar beet protox-1 cDNA obtained, designated “sugar beetprotox-1”, is full-length based on comparison with the other known plantprotox peptide sequences (Table 1A). Sugar beet protox-1 is 1910-bp inlength and encodes a protein of 60 kDa. The nucleotide sequence of thiscDNA and the amino acid sequence it encodes are set forth in SEQ IDNOs:15 and 16, respectively. The sugar beet protein is 73% identical(82% similar) to the Arabidopsis protox-1 protein.

[0476] Sugar beet protox-1, in the pBluescript SK vector, was depositedJul. 29, 1996, as pWDC-16 (NRRL #B-21595N).

Example 5 Isolation of an Oilseed Rape Protox-1 cDNA Based on SequenceHomology to an Arabidopsis Protox-1 Coding Sequence

[0477] A Lambda Uni-Zap II cDNA library prepared from Brassica napus(3-4 wk. mature green leaves) was obtained from Dr. Guenther Ochs,Institut Fuer Allgemeine Botanik, Johannes Gutenberg-Universitaet Mainz,Germany (Günther Ochs, Gerald Schock, and Aloysius Wild, Plant Physiol.103: 303-304 (1993)). Approximately 50,000 pfu of the cDNA library wereplated at a density of approximately 5,000 pfu per 10 cm Petri dish andduplicate filter lifts were made onto nitrocellulose membranes(Schleicher and Schuell). The plaque lifts were probed with theArabidopsis protox-1 cDNA (SEQ ID NO:1) labeled with 32P-dCTP by therandom priming method (Life Technologies). Hybridization conditions were7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C.Wash conditions were 2×SSC, 1% SDS at 50° C. (Church and Gilbert(1984)). Positively hybridizing plaques were purified and in vivoexcised into pBluescript plasmids. The sequences of the cDNA insertswere determined by the chain termination method using dideoxyterminators labeled with fluorescent dyes (Applied Biosystems, Inc.).The longest oilseed rape protox-1 cDNA obtained, designated “rapeprotox-1”, is full-length based on comparison with the other known plantprotox peptide sequences (Table 1A). Rape protox-1 is 1784-bp in lengthand encodes a protein of 57.3kD. The nucleotide sequence of this cDNAand the amino acid sequence it encodes are set forth in SEQ ID NOs:17and 18, respectively. The oilseed rape protein is 87% identical (92%similar) to the Arabidopsis protox-1 protein.

[0478] Rape protox-1, in the pBluescript SK vector, was deposited August23, 1996, as pWDC-17 (NRRL#B-21615).

Example 6 Isolation of a Rice Protox-1 cDNA Based on Sequence Homologyto a Maize Protox-1 Coding Sequence

[0479] A Lambda gtl 1 cDNA library prepared from Oryza sativa (5 dayetiolated shoots) was purchased from Clontech. Approximately 50,000 pfuof the cDNA library were plated at a density of approximately 5,000 pfuper 10 cm Petri dish and duplicate filter lifts were made ontonitrocellulose membranes (Schleicher and Schuell). The plaque lifts wereprobed with the maize protox-1 cDNA (SEQ ID NO:5) labeled with 32P-dCTPby the random priming method (Life Technologies). Hybridizationconditions were 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1mM EDTA at 50° C. Wash conditions were 2×SSC, 1% SDS at 50° C. (Churchand Gilbert (1984)). Positively hybridizing plaques were purified, andlambda DNA was prepared using the Wizard Lambda-Prep kit (Promega). ThecDNA inserts were subcloned as EcoRI fragments into the pBluescript SKvector using standard techniques. The sequences of the cDNA inserts weredetermined by the chain termination method using dideoxy terminatorslabeled with fluorescent dyes (Applied Biosystems, Inc.). The longestrice protox-1 cDNA obtained, designated “rice protox-1”, was 1224-bp inlength. Rice protox-1 lacks coding sequence for the transit peptide plusapproximately 172 amino acids of the mature coding sequence based oncomparison with the other known plant protox peptide sequences (Table1A). The nucleotide sequence of this partial cDNA and the amino acidsequence it encodes are set forth in SEQ ID NOs:19 and 20, respectively.

[0480] Rice protox-1, in the pBluescript SK vector, was deposited Dec.6, 1996, as pWDC-18 (NRRL #B-21648).

Example 7 Isolation of a Sorghum Protox-l cDNA Based on SequenceHomology to a Maize Protox-1 Coding Sequence

[0481] A Lambda-Zap II cDNA library prepared from Sorghum bicolor (3-6day green seedlings) was obtained from Dr. Klaus Pfizenmaier, Instituteof Cell Biology and Immunology, University of Stuttgart, Germany (HaraldWajant, Karl-Wolfgang Mundry, and Klaus Pfizenmaier, Plant Mol. Biol.26: 735-746 (1994)). Approximately 50,000 pfu of the cDNA library wereplated at a density of approximately 5,000 pfu per 10 cm Petri dish andduplicate filter lifts were made onto nitrocellulose membranes(Schleicher and Schuell). The plaque lifts were probed with the maizeprotox-1 cDNA (SEQ ID NO:5) labeled with 32P-dCTP by the random primingmethod (Life Technologies). Hybridization conditions were 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C. Washconditions were 2×SSC, 1% SDS at 50° C. (Church and Gilbert (1984)).Positively hybridizing plaques were purified and in vivo excised intopBluescript plasmids. The sequences of the cDNA inserts were determinedby the chain termination method using dideoxy terminators labeled withfluorescent dyes (Applied Biosystems, Inc.). The longest sorghumprotox-1 cDNA obtained, designated “sorghum protox-1”, was 1590-bp inlength. Sorghum protox-1 lacks coding sequence for the transit peptideplus approximately 44 amino acids of the mature coding sequence based oncomparison with the other known plant protox peptide sequences (Table1A). The nucleotide sequence of this partial cDNA and the amino acidsequence it encodes are set forth in SEQ ID NOs:21 and 22, respectively.

[0482] Sorghum protox-1, in the pBluescript SK vector, was depositedDec. 6, 1996, as pWDC-19 (NRRL #B-21649).

Example 8 Isolation of a Sugar Cane Protox-1 cDNA Based on SequenceHomology to a Maize Protox-1 Coding Sequence

[0483] A Lambda-Zap II cDNA library prepared from sugar cane wasobtained from Henrik Albert of USDA/ARS at the Hawaii AgriculturalResearch Center. Approximately 50,000 pfu of the cDNA library wereplated at a density of approximately 5,000 pfu per 10 cm Petri dish andduplicate filter lifts were made onto nitrocellulose membranes(Schleicher and Schuell). The plaque lifts were probed with the maizeprotox-1 cDNA (SEQ ID NO:5) labeled with 32P-dCTP by the random primingmethod (Life Technologies). Hybridization conditions were 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 50° C. Washconditions were 2X SSC, 1% SDS at 50° C. (Church and Gilbert (1984)).Positively hybridizing plaques were purified and in vivo excised intopBluescript plasmids. The sequences of the cDNA inserts were determinedby the chain termination method using dideoxy terminators labeled withfluorescent dyes (Applied Biosystems, Inc.). The longest sugar caneprotox-1 cDNA obtained, designated “sugar cane protox-1”, was 633-bp inlength. Sugar cane protox-1 lacks coding sequence for the transitpeptide plus approximately 382 amino acids of the mature coding sequencebased on comparison with the other known plant protox peptide sequences(Table 1A). The nucleotide sequence of this partial cDNA and the aminoacid sequence it encodes are set forth in SEQ ID NOs:36 and 37,respectively.

Example 9 Demonstration of Plant Protox Clone Sensitivity to ProtoxInhibitory Herbicides in a Bacterial System

[0484] Liquid cultures of protox-1/SASX38, protox-2/SASX38 andpBluescript/XL1-Blue were grown in L amp¹⁰⁰. One hundred microliteraliquots of each culture were plated on L amp¹⁰⁰ media containingvarious concentrations (1.0 nM-10 mM) of a protox inhibitory aryluracilherbicide of formula XVII. Duplicate sets of plates were incubated for18 hours at 37° C.

[0485] The protox⁺ E. coli strain XL1-Blue showed no sensitivity to theherbicide at any concentration, consistent with reported resistance ofthe native bacterial enzyme to similar herbicides. The protox-1/SASX38was clearly sensitive, with the lawn of bacteria almost entirelyeliminated by inhibitor concentrations as low as 10 nM. Theprotox-2/SASX38 was also sensitive, but only at a higher concentration(10 μM) of the herbicide. The herbicide was effective even on platesmaintained almost entirely in the dark. The toxicity of the herbicidewas entirely eliminated by the addition of 20 μg/ml hematin to theplates.

[0486] The different herbicide tolerance between the two plant protoxstrains is likely the result of differential expression from these twoplasmids, rather than any inherent difference in enzyme sensitivity.Protox-1/SASX38 grows much more slowly than protox-2/SASX38 in anyheme-deficient media. In addition, the Mzprotox-2/SASX38 strain, with agrowth rate comparable to Arab protox-1/SASX38, is also very sensitiveto herbicide at the lower (10-100 nM) concentrations.

Section B: Identification and Characterization of Plant Protox GenesResistant to Protox-Inhibitory Herbicides Example 10 Selecting for PlantProtox Genes Resistant to Protox-Inhibitory Herbicides in the E. coliExpression System

[0487] An Arabidopsis thaliana (Landsberg) cDNA library in the plasmidvector pFL61 (Minet et al., Plant J. 2:417-422 (1992) was obtained andamplified. The E. coli hemG mutant SASX38 (Sasarman et al., J. Gen.Microbiol. 113:297(1979)) was obtained and maintained on L mediacontaining 20 ug/ml hematin (United States Biochemicals). The plasmidlibrary was transformed into SASX38 by electroporation using the Bio-RadGene Pulser and the manufacturer's conditions. The electroporated cellswere plated on L agar containing 100ug/ml ampicillin at a density ofapproximately 500,000 transformants/10 cm plate. The cells were thenincubated at 37° C. for 40 hours in low light and selected for theability to grow without the addition of exogenous heme. Heme prototrophswere recovered at a frequency of 400/10⁷ from the pFL61 library.Sequence analysis of twenty-two complementing clones showed that nineare of the type designated “protox-1,” the protox gene expected toexpress a chloroplastic protox enzyme.

[0488] The pFL61 library is a yeast expression library, with theArabidopsis cDNAs inserted bidirectionally. These cDNAs can also beexpressed in bacteria. The protox cDNAs apparently initiate at anin-frame ATG in the yeast PGK 3′ sequence approximately 10 amino acids5′ to the NotI cloning site in the vector and are expressed either fromthe lacZ promoter 300bp further upstream or from an undefined crypticbacterial promoter. Because protox-1 cDNAs that included significantportions of a chloroplast transit sequence inhibited the growth of theE. coli SASX38 strain, the clone with the least amount of chloroplasttransit sequence attached was chosen for mutagenesis/herbicide selectionexperiments. This clone, pSLV19, contains only 17 amino acids of theputative chloroplast transit peptide, with the DNA sequence beginning atbp-151 of the Arabidopsis protox-1 cDNA (SEQ ID NO:1).

[0489] The plasmid pSLV19 was transformed into the random mutagenesisstrain XL1-Red (Stratagene, La Jolla, Calif.). The transformation wasplated on L media containing 50ug/ml ampicillin and incubated for 48hours at 37° C. Lawns of transformed cells were scraped from the platesand plasmid DNA prepared using the Wizard Megaprep kit (Promega,Madison, Wis.). Plasmid DNA isolated from this mutator strain ispredicted to contain approximately one random base change per 2000nucleotides (see Greener et al., Strategies 7(2):32-34 (1994).

[0490] The mutated plasmid DNA was transformed into the hemG mutantSASX38 (Sasarman et al., J. Gen. Microbiol. 113:297 (1979) and plated onL media containing various concentrations of protox-inhibiting herbicide(formula XVII). The plates were incubated for 2 days at 37° C. PlasmidDNA was isolated from all colonies that grew in the presence ofherbicide concentrations that effectively killed the wild type strain.The isolated DNA was then transformed into SASX38 and plated again onherbicide to ensure that the resistance observed was plasmid-bome. Theprotox coding sequence from plasmids passing this screen was excised byNotI digestion, recloned into an unmutagenized vector, and tested againfor the ability to confer herbicide tolerance. The DNA sequence ofprotox cDNAs that conferred herbicide resistance was then determined andmutations identified by comparison with the wild type Arabidopsisprotox-1 sequence (SEQ ID NO:1).

[0491] A single coding sequence mutant was recovered from the firstmutagenesis experiment. This mutant leads to enhanced herbicide“resistance” only by increasing growth rate. It contains a C to Amutation at nucleotide 197 in SEQ ID NO:1 in the truncated chloroplasttransit sequence of pSLV19, converting an ACG codon for threonine to anAAG codon for lysine at amino acid 56 of SEQ ID NO:2, and resulting inbetter complementation of the bacterial mutant. This plasmid alsocontains a silent coding sequence mutation at nucleotide 1059, with AGT(Ser) changing to AGC (Ser). This plasmid was designated pMut-1.

[0492] The pMut-1 plasmid was then transformed into the mutator XL1-Redstrain as described above and the mutated DNA was isolated and plated onan herbicide concentration that is lethal to the unmutagenized pMut-1protox gene. Herbicide tolerant colonies were isolated after two days at37° C. and analyzed as described above. Multiple plasmids were shown tocontain herbicide resistant protox coding sequences. Sequence analysisindicated that the resistant genes fell into two classes. One resistancemutation identified was a C to T change at nucleotide 689 in theArabidopsis protox-1 sequence set forth in SEQ ID NO:1. This changeconverts a GCT codon for alanine at amino acid 220 of SEQ ID NO:2 to aGTT codon for valine, and was designated pAraC-1Val (see, Table 1B;sub-sequence 3).

[0493] A second class of herbicide resistant mutant contains an A to Gchange at nucleotide 1307 in the Arabidopsis protox-1 sequence. Thischange converts a TAC codon for tyrosine at amino acid 426 to a TGCcodon for cysteine, and was designated pAraC-2Cys (see, Table 1B;sub-sequence 7).

[0494] A third resistant mutant has a G to A change at nucleotide 691 inthe Arabidopsis protox-1 sequence. This mutation converts a GGT codonfor glycine at amino acid 221 to an AGT codon for serine at the codonposition adjacent to the mutation in pAraC-1. This plasmid wasdesignated pAraC-3Ser (see, Table 1B; sub-sequence 4).

[0495] Resistant mutant pAraC-2Cys, in the pMut-1 plasmid, was depositedon Nov. 14, 1994 under the designation pWDC-7 with the AgriculturalResearch Culture Collection and given the deposit designation NRRL#21339N.

Example 11 Additional Herbicide-Resistant Codon Substitutions atPositions Identified in the Random Screen

[0496] The amino acids identified as herbicide resistance sites in therandom screen are replaced by other amino acids and tested for functionand for herbicide tolerance in the bacterial system.Oligonucleotide-directed mutagenesis of the Arabidopsis protox-1sequence is performed using the Transformer Site-Directed MutagenesisKit (Clontech, Palo Alto, Calif.). After amino acid changes areconfirmed by sequence analysis, the mutated plasmids are transformedinto SASX38 and plated on L-amp¹⁰⁰ media to test for function and onvarious concentrations of protox-inhibiting herbicide to test fortolerance.

[0497] This procedure is applied to the alanine codon at nucleotides688-690 and to the tyrosine codon at nucleotides 1306-1308 of theArabidopsis protox-1 sequence (SEQ ID NO:1). The results demonstratethat the alanine codon at nucleotides 688-690 can be changed to a codonfor valine (pAraC-lVal), threonine (pAraC-1Thr), leucine (pAraC-1Leu),cysteine (pAraC-1Cys), or isoleucine (pAraC-1Ile) to yield anherbicide-resistant protox enzyme that retains function (see, Table 1B;sub-sequence 3). The results further demonstrate that the tyrosine codonat nucleotides 1306-1308 can be changed to a codon for cysteine(pAraC-2Cys), isoleucine (pAraC-2Ile), leucine (pAraC-2Leu), threonine(pAraC-2Thr), methionine (pAraC-2Met), valine (pAraC-2Val), or alanine(pAraC-2Ala) to yield an herbicide-resistant protox enzyme that retainsfunction (see, Table 1B; sub-sequence 7).

Example 12 Isolation of Additional Mutations that Increase EnzymeFunction and/or Herbicide Tolerance of Previously Identified ResistantMutants

[0498] Plasmids containing herbicide resistant protox genes aretransformed into the mutator strain XL1-Red and mutated DNA is isolatedas described above. The mutated plasmids are transformed into SASX38 andthe transformants are screened on herbicide concentrations (formulaXVII) sufficient to inhibit growth of the original “resistant” mutant.Tolerant colonies are isolated and the higher tolerance phenotype isverified as being coding sequence dependent as described above. Thesequence of these mutants is determined and mutations identified bycomparison to the progenitor sequence.

[0499] This procedure was applied to the pAraC-lVal mutant describedabove. The results demonstrate that the serine codon at amino acid 305(SEQ ID NO:2) can be changed to a codon for leucine to yield an enzymewith higher tolerance to protox-inhibiting herbicides than thepAraC-1Val mutant alone. This second site mutation is designatedAraC305Leu (see, Table 1B; sub-sequence 13). The same results aredemonstrated for the threonine codon at amino acid 249, where a changeto either isoleucine or to alanine leads to a more tolerant enzyme (see,Table 1B; sub-sequence 12). These changes are designated AraC249Ile andAraC249Ala, respectively.

[0500] The procedure was also applied to the pAraC-2Cys mutant describedabove. The results demonstrate that the proline codon at amino acid 118(SEQ ID NO:2) can be changed to a codon for leucine to yield an enzymewith higher tolerance to protox-inhibiting herbicides than thepAraC-1Cys mutant alone. This mutation is designated AraC118Leu (see,Table 1B; sub-sequence 11). The same results are demonstrated for theserine codon at amino acid 305, where a change to leucine leads to amore tolerant pAraC-2Cys enzyme (see, Table 1B; sub-sequence 13). Thischange was also isolated with the pAraC-1Val mutant as described aboveand is designated AraC305Leu. Additional mutations that enhance theherbicide resistance of the pAraC-2Cys mutant include an asparagine toserine change at amino acid 425, designated AraC425Ser (Table 1B;sub-sequence 14), and a tyrosine to cysteine at amino acid 498,designated AraC498Cys (Table 1B; sub-sequence 15).

[0501] These changes (Table 1B; sub-sequences 11-15) are referred to as“second site” mutations, because they are not sufficient to conferherbicide tolerance alone, but rather enhance the function and/or theherbicide tolerance of an already mutant enzyme. This does not precludethe possibility that other amino acid substitutions at these sites couldsuffice to produce an herbicide tolerant enzyme since exhaustive testingof all possible replacements has not been performed.

Example 13 Combining Identified Resistance Mutations with IdentifiedSecond Site Mutations to Create Highly Functional/Highly Tolerant ProtoxEnzymes

[0502] The AraC305Leu mutation described above was found to enhance thefunction/herbicide resistance of both the AraC-1Val and the AraC-2Cysmutant plasmids. In an effort to test the general usefulness of thissecond site mutation, it was combined with the AraC-2Leu, AraC-2Val, andAraC-2Ile mutations and tested for herbicide tolerance. In each case,the AraC305Leu change significantly increased the growth rate of theresistant protox mutant on protox-inhibiting herbicide. Combinations ofthe AraC-2Ile resistant mutant with either the second site mutantAraC249Ile or AraC118Leu also produced more highly tolerant mutantprotox enzymes. The AraC249Ile mutation demonstrates that a second sitemutation identified as enhancing an AraC-1 (sub-sequence 3) mutant mayalso increase the resistance of an AraC-2 (sub-sequence 7) mutant. Athree mutation plasmid containing AraC-2Ile, AraC305Leu, and AraC249Ile(Table 1B; sub-sequences 7, 13, and 12) has also been shown to produce ahighly functional, highly herbicide tolerant protox-1 enzyme.

Example 14 Identification of Sites in the Maize Protox-1 Gene that CanBe Mutated to Give Herbicide Tolerance

[0503] The pMut-1 Arabidopsis protox -1 plasmid described above is veryeffective when used in mutagenesis/screening experiments in that itgives a high frequency of genuine coding sequence mutants, as opposed tothe frequent up-promoter mutants that are isolated when other plasmidsare used. In an effort to create an efficient plasmid screening systemfor maize protox-1, the maize cDNA was engineered into the pMut-1 vectorin approximately the same sequence context as the Arabidopsis cDNA.Using standard methods of overlapping PCR fusion, the 5 end of thepMut-1 Arabidopsis clone (including 17 amino acids of chloroplasttransit peptide with one mis-sense mutation as described above) wasfused to the maize protox-1 cDNA sequence starting at amino acid number14 of the maize sequence (SEQ ID NO:6). The 3′ end of the maize cDNA wasunchanged. NotI restriction sites were placed on both ends of thisfusion, and the chimeric gene was cloned into the pFL61 plasmid backbonefrom pMut-1. Sequence analysis revealed a single nucleotide PCR-derivedsilent mutation that converts the ACG codon at nucleotides 745-747 (SEQID NO:5) to an ACT codon, both of which encode threonine. This chimericArab-maize protox-1 plasmid was designated pMut-3.

[0504] The pMut-3 plasmid was transformed into the mutator XL1 -Redstrain as described above and the mutated DNA was isolated and plated ona herbicide concentration (formula XVII) that was lethal to theunmutagenized pMut-3 maize protox gene. Herbicide tolerant colonies wereisolated after two days at 37° C. and analyzed as described above. Thisanalysis revealed multiple plasmids containing herbicide resistantprotox coding sequences. Sequence analysis showed 5 single base changesthat individually result in an herbicide tolerant maize protox-1 enzyme.Three of these mutations correspond to amino acid changes previouslyshown to confer tolerance at the homologous position in the Arabidopsisprotox-1 gene. Two of the three are pMzC-1Val and pMzC-1Thr, convertingthe alanine (GCT) at amino acid 164 (SEQ ID NO:6) to either valine (GAT)or to threonine (ACT). This position corresponds to the pAraC-1mutations described above (see, Table 1B; sub-sequence 3). The thirdanalogous change, pMzC-3Ser, converts the glycine (GGT) at amino acid165 to Serine (AGT), corresponding to the AraC-3Ser mutation describedabove (see, Table 1B; sub-sequence 4). These results serve to validatethe expectation that herbicide-tolerant mutations identified in oneplant protox gene may also confer herbicide tolerance in an equivalentplant protox gene from another species.

[0505] Two of the mutations isolated from the maize protox-1 screenresult in amino acid changes at residues not previously identified asherbicide resistance sites. One change (Mz159Phe) converts cysteine(TGC) to phenylalanine (TTC) at amino acid 159 of the maize protox-1sequence (SEQ ID NO:6) (see, Table 1B; sub-sequence 2). The second(Mz419Thr) converts isoleucine (ATA) to threonine (ACA) at amino acid419 (see, Table 1B; sub-sequence 9).

[0506] Additional amino acid substitutions were made and tested at threeof the maize mutant sites. Tolerance was demonstrated when glycine 165was changed to leucine (pMzC-3Leu) or when cysteine 159 was changed toeither leucine (Mz159Leu) or to lysine (Mz159Lys) (see, Table 1B;sub-sequences 4 and 2). Tolerant enzymes were also created by changingisoleucine 419 to histidine (Mz419His), glycine (Mz419Gly), orasparagine (Mz419Asn) (see, Table 1B; sub-sequence 9).

[0507] Individual amino acid changes that produced highly herbicidetolerant Arabidopsis protox-1 enzymes were engineered into the maizeprotox-1 gene by site-directed mutagenesis as described above. Bacterialtesting demonstrated that changing the alanine (GCT) at amino acid 164(SEQ ID NO:6) to leucine (CTT) produced a highly tolerant maize enzyme(pMzC-1Leu) (see, Table 1B; sub-sequence 3). No mutation analogous tothe AraC-2 site (Table 1B; sub-sequence 7) in Arabidopsis was isolatedin the maize random screen. However, changing this site, tyrosine 370 inthe maize enzyme (SEQ ID NO:6), to either isoleucine (pMzC-2Ile) ormethionine (pMzC-2Met) did produce herbicide tolerant enzymes (see,Table 1B; sub-sequence 7).

[0508] Additional mutant screens performed as described earlier in thisexample, except using formulas XXIIIa and XXIIIB instead of XVII,identified three additional amino acid changes that confer tolerantprotox enzymes. One, using formula XXIIIb, demonstrated that changingthe arginine (CGT) at amino acid 88 (SEQ ID NO:6) to cysteine (TGT)produced a highly tolerant maize enzyme (Mz88Cys) (see, Table 1B;sub-sequence 1). Another, using formula XXIIIa, demonstrated thatchanging both the leucine (TTA) at amino acid 347 (SEQ ID NO:6) toserine (TCA) and the alanine (GCA) at amino acid 453 (SEQ ID NO:6) tothreonine (ACA) produced a highly tolerant maize enzyme (Mz347Ser453Thr)(see, Table 1B; sub-sequences 16 and 17). Unlike the second sitemutations described above, which increase enzyme function and/orherbicide tolerance of previously identified resistant mutants,Mz347Ser453Thr is a “double mutant” that requires that both mutations bepresent for herbicide tolerance.

Example 15 Identification of Sites in the Wheat Protox-1 Gene that canbe Mutated to Give Herbicide Tolerance

[0509] To create an efficient plasmid screening system for wheatprotox-1, the wheat cDNA was engineered into the pMut-1 vector asdescribed above for the maize cDNA. This chimeric Arab-wheat protox-1plasmid is designated pMut-4. The pMut-4 DNA was mutated and screenedfor herbicide tolerance as described above. This analysis revealedmultiple plasmids containing herbicide resistant protox codingsequences. Sequence analysis showed 7 single base changes thatindividually result in an herbicide tolerant wheat protox-1 enzyme. Fourof these mutations correspond to amino acid changes previously shown toconfer tolerance at the homologous position in the Arabidopsis and/or inthe maize protox-1 gene. Two, pWhtC-1Val and pWhtC-1Thr, convert thealanine (GCT) at amino acid 211 (SEQ ID NO:10) to valine (GAT) and tothreonine (ACT), respectively. This position corresponds to the pAraC-1mutations described above (see, Table 1B; sub-sequence 3). The thirdanalogous change, pWhtC-3Ser, converts the glycine (GGT) at amino acid212 to serine (AGT), corresponding to the AraC-3Ser mutation describedabove (see, Table 1B; sub-sequence 4). The fourth, Wht466Thr, convertsisoleucine (ATA) to threonine (ACA) at amino acid 466, corresponding tothe Mz419Thr mutant from maize (see, Table 1B; sub-sequence 9).

[0510] Three of the mutations isolated from the wheat protox-1 screenresult in amino acid changes at residues not previously identified asherbicide resistance sites. One change (Wht356Leu) converts valine (GTT)to leucine (CTT) at amino acid 356 of the wheat protox-1 sequence (SEQID NO:10) (see, Table 1B; sub-sequence 6). A second (Wht421Pro) convertsserine (TCT) to proline (CCT) at amino acid 421 (see, Table 1B;sub-sequence 8). The third (Wht502Ala) converts valine (GTT) to alanine(GCT) at amino acid 502 (see, Table 1B; sub-sequence 10).

Example 16 Identification of Sites in the Soybean Protox-1 Gene that canbe Mutated to Give Herbicide Tolerance

[0511] To create an efficient plasmid screening system for soybeanprotox-1, the soybean cDNA was engineered into the pMut-1 vector asdescribed above for the maize cDNA. This chimeric Arab-soybean protox-1plasmid is designated pMut-5. The pMut-5 DNA was mutated and screenedfor herbicide tolerance as described above. This analysis revealedmultiple plasmids containing herbicide resistant protox codingsequences. Sequence analysis showed 4 single base changes thatindividually result in an herbicide tolerant soybean protox-1 enzyme.Two of these mutations correspond to amino acid changes previously shownto confer tolerance at the homologous position in the Arabidopsis and/orin the wheat protox-1 gene. One, pSoyC-1Thr, converts the alanine (GCA)at amino acid 226 (SEQ ID NO:12) to threonine (ACA). This positioncorresponds to the pAraC-1Thr mutation described above (see, Table 1B;sub-sequence 3). The second analogous change, Soy517Ala, converts thevaline (GTT) at amino acid 517 to alanine (GCT), corresponding to theWht502Ala mutation from wheat (see, Table 1B; sub-sequence 10).

[0512] Two of the mutations isolated from the soybean protox-1 screenresult in amino acid changes at a residue not previously identified asan herbicide resistance site. One change (Soy369Ser) converts proline(CCT) to serine (TCT) at amino acid 369 of the soybean protox-1 sequence(SEQ ID NO:12) (see, Table 1B; sub-sequence 5). A second (Soy369His)converts this same proline369 to histidine (CAT) (see, Table 1B;sub-sequence 5).

[0513] Individual amino acid changes that produced highly herbicidetolerant Arabidopsis protox-1 enzymes were engineered into the soybeanprotox-1 gene by site directed mutagenesis as described above. Bacterialtesting demonstrated that changing the alanine (GCA) at amino acid 226(SEQ ID NO:12) to leucine (pSoyC-1Leu) produced a tolerant soybeanenzyme (see, Table 1B; sub-sequence 3). Changing the tyrosine (TAC) atamino acid 432 (SEQ ID NO:12) to either leucine (pSoyC-2Leu) orisoleucine (pSoyC-2Ile) also produced herbicide tolerant enzymes (see,Table 1B; sub-sequence 7).

Example 17 Identification of Sites in the Sugar Beet Protox-1 Gene thatcan be Mutated to Give Herbicide Tolerance

[0514] To create an efficient plasmid screening system for sugar beetprotox-1, the sugar beet cDNA was engineered into the pMut-1 vector asdescribed above for the maize cDNA. This chimeric Arab-sugar beetprotox-1 plasmid is designated pMut-6. The pMut-6 DNA was mutated andscreened for herbicide tolerance as described above. This analysisrevealed multiple plasmids containing herbicide resistant protox codingsequences. Sequence analysis showed a single base change that results inan herbicide tolerant sugar beet protox-1 enzyme. This change(pSugC-2Cys) converts tyrosine (TAC) at amino acid 449 to cysteine (TGC)and is analogous to the AraC-2 mutations in Arabidopsis (see, Table 1B;sub-sequence 7).

[0515] Individual amino acid changes that produced highly herbicidetolerant Arabidopsis protox-1 enzymes were engineered into the sugarbeet protox-1 gene by site directed mutagenesis as described above.Bacterial testing demonstrated that changing the tyrosine (TAC) at aminoacid 449 to leucine (pSugC-2Leu), isoleucine (pSugC-2Ile), valine(pSugC-2Val), or methionine (pSugC-2Met) produced herbicide tolerantsugar beet enzymes (see, Table 1B; sub-sequence 7).

Example 18 Identification of Sites in the Cotton Protox-1 Gene that canbe Mutated to Give Herbicide Tolerance

[0516] In an effort to create an efficient plasmid screening system forcotton protox-1, the cotton cDNA was engineered into the pMut-1 vectoras described above for the maize cDNA. This chimeric Arab-cottonprotox-1 plasmid is designated pMut-7. The pMut-7 DNA was mutated andscreened for herbicide tolerance as described above. This analysisrevealed multiple plasmids containing herbicide resistant protox codingsequences. Sequence analysis showed 3 single base changes thatindividually result in an herbicide tolerant cotton protox-1 enzyme. Twomutants, pCotC-2Cys and pCotC-2Arg, change tyrosine (TAC) at amino acid428 (SEQ ID NO:16) to cysteine (TGC) and to arginine (CGC), respectively(see, Table 1B; sub-sequence 7). Arginine is a novel substitution givingtolerance at this previously identified AraC-2 (sub-sequence 7) site.The third mutation (Cot365Ser) converts proline (CCC) to serine (TCC) atamino acid 365. This change corresponds to the soybean mutant Soy369Ser(see, Table 1B; sub-sequence 5).

Example 19 Demonstration of Resistant Mutations' Cross-Tolerance toVarious Protox-Inhibiting Compounds

[0517] Resistant mutant plasmids, originally identified based onresistance against a single protox inhibitory herbicide, were testedagainst a spectrum of other protox inhibiting compounds. For this test,the SASX38 strain containing the wild-type plasmid is plated on a rangeof concentrations of each compound to determine the lethal concentrationfor each one. Resistant mutant plasmids in SASX38 are plated and scoredfor the ability to survive on a concentration of each compound at least10 fold higher than the concentration that is lethal to the SASX38strain containing the wild-type plasmid.

[0518] Results from bacterial cross-tolerance testing, illustrated inTables 3A and 3B, show that each of the mutations identified conferstolerance to a variety of protox inhibiting compounds.

Section C: Expression of Herbicide-Resistant Protox Genes in TransgenicPlants Example 20 Engineering of Plants Tolerant to Protox-InhibitingHerbicides by Homologous Recombination or Gene Conversion

[0519] Because the described mutant coding sequences effectively conferherbicide tolerance when expressed under the control of the nativeprotox promoter, targeted changes to the protox coding sequence in itsnative chromosomal location represent an alternative means forgenerating herbicide tolerant plants and plant cells. A fragment ofprotox DNA containing the desired mutations, but lacking its ownexpression signals (either promoter or 3′ untranslated region) can beintroduced by any of several art-recognized methods (for instance,Agrobacterium transformation, direct gene transfer to protoplasts,microprojectile bombardment), and herbicide-tolerant transformantsselected. The introduced DNA fragment also contains a diagnosticrestriction enzyme site or other sequence polymorphism that isintroduced by site-directed mutagenesis in vitro without changing theencoded amino acid sequence (i.e. a silent mutation). As has beenpreviously reported for various selectable marker and herbicidetolerance genes (see, e.g., Paszkowski et al., EMBO J. 7: 4021-4026(1988); Lee et al., Plant Cell 2: 415-425 (1990); Risseeuw et al., PlantJ. 7: 109-119 (1995)). some transformants are found to result fromhomologous integration of the mutant DNA into the protox chromosomallocus, or from conversion of the native protox chromosomal sequence tothe introduced mutant sequence. These transformants are recognized bythe combination of their herbicide-tolerant phenotype, and the presenceof the diagnostic restriction enzyme site in their protox chromosomallocus.

Example 21 Construction of Plant Transformation Vectors

[0520] Numerous transformation vectors are available for planttransformation, and the genes of this invention can be used inconjunction with any such vectors. The selection of vector for use willdepend upon the preferred transformation technique and the targetspecies for transformation. For certain target species, differentantibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformation include the nptII gene, whichconfers resistance to kanamycin and related antibiotics (Messing &Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187(1983)), the bar gene, which confers resistance to the herbicidephosphinothricin (White et al., Nucl Acids Res 18: 1062 (1990), Spenceret al. Theor Appl Genet 79: 625-631(1990)), the hph gene, which confersresistance to the antibiotic hygromycin (Blochinger & Diggelmann, MolCell Biol 4: 2929-2931), and the dhfr gene, which confers resistance tomethotrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)).

[0521] I. Construction of Vectors Suitable for AgrobacteriumTransformation

[0522] Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) andpXYZ. Below the construction of two typical vectors is described.

[0523] Construction of pCIB200 and pCIB2001: The binary vectors pCIB200and pCIB2001 are used for the construction of recombinant vectors foruse with Agrobacterium and was constructed in the following manner.pTJS75kan was created by NarI digestion of pTJS75 (Schmidhauser &Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of thetetracycline-resistance gene, followed by insertion of an Acci fragmentfrom pUC4K carrying an NPTII (Messing & Vierra, Gene 19: 259-268 (1982);Bevan et al., Nature 304: 184-187 (1983); McBride et al., PlantMolecular Biology 14: 266-276 (1990)). XhoI linkers were ligated to theEcoRV fragment of pCIB7, which contains the left and right T-DNAborders, a plant selectable nos/nptII chimeric gene and the pUCpolylinker (Rothstein et al., Gene 53: 153-161 (1987)), and theXhoI-digested fragment was cloned into SalI-digested pTJS75kan to createpCIB200 (see also EP 0 332 104, example 19). pCIB200 contains thefollowing unique polylinker restriction sites: EcoRI, SstI, KpnI, BglII,XbaI, and SalI. pCIB2001 is a derivative of pCIB200, which is created bythe insertion into the polylinker of additional restriction sites.Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl,KpnI, BglII, Xbal, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI.pCIB2001, in addition to containing these unique restriction sites alsohas plant and bacterial kanamycin selection, left and right T-DNAborders for Agrobacterium-mediated transformation, the RK2-derived trfAfunction for mobilization between E. coli and other hosts, and the OriTand OriV functions also from RK2. The pCIB2001 polylinker is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

[0524] Construction of pCIB 10 and Hygromycin Selection DerivativesThereof: The binary vector pCIB 10 contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al., Gene 53: 153-161 (1987).Various derivatives of pCIB10 have been constructed that incorporate thegene for hygromycin B phosphotransferase described by Gritz et al., Gene25: 179-188 (1983)). These derivatives enable selection of transgenicplant cells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

[0525] II. Construction of Vectors Suitable for non-AgrobacteriumTransformation.

[0526] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovethat contain T-DNA sequences. Transformation techniques that do not relyon Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g. PEG and electroporation) and microinjection. Thechoice of vector depends largely on the preferred selection for thespecies being transformed. Below, the construction of some typicalvectors is described.

[0527] Construction of pCIB3064: pCIB3064 is a pUC-derived vectorsuitable for direct gene transfer techniques in combination withselection by the herbicide basta (or phosphinothricin). The plasmidpCIB246 comprises the CaMV 35S promoter in operational fusion to the E.coli GUS gene and the CaMV 35S transcriptional terminator and isdescribed in the PCT published application WO 93/07278. The 35S promoterof this vector contains two ATG sequences 5′ of the start site. Thesesites were mutated using standard PCR techniques in such a way as toremove the ATG's and generate the restriction sites SspI and PvuII. Thenew restriction sites were 96 and 37-bp away from the unique SalI siteand 101 and 42-bp away from the actual start site. The resultantderivative of pCIB246 was designated pCIB3025. The GUS gene was thenexcised from pCIB3025 by digestion with SalI and SacI, the terminirendered blunt and religated to generate plasmid pCIB3060. The plasmidpJIT82 was obtained from the John Innes Centre, Norwich and the a 400-bpSmaI fragment containing the bar gene from Streptomycesviridochromogenes was excised and inserted into the HpaI site ofpCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generatedpCIB3064, which comprises the bar gene under the control of the CaMV 35Spromoter and terminator for herbicide selection, a gene for ampicillinresistance (for selection in E. coli) and a polylinker with the uniquesites SphI, PstI, HindIII, and BamHI. This vector is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

[0528] Construction of pSOG19 and pSOG35: pSOG35 is a transformationvector that utilizes the E. coli gene dihydrofolate reductase (DHFR) asa selectable marker conferring resistance to methotrexate. PCR was usedto amplify the 35S promoter (˜800-bp), intron 6 from the maize Adhl gene(˜550-bp) and 18-bp of the GUS untranslated leader sequence from pSOG10.A 250-bp fragment encoding the E. coli dihydrofolate reductase type IIgene was also amplified by PCR and these two PCR fragments wereassembled with a SacI-PstI fragment from pBI221 (Clontech), whichcomprised the pUC19 vector backbone and the nopaline synthaseterminator. Assembly of these fragments generated pSOG19, which containsthe 35S promoter in fusion with the intron 6 sequence, the GUS leader,the DHFR gene and the nopaline synthase terminator. Replacement of theGUS leader in pSOG19 with the leader sequence from Maize ChloroticMottle Virus (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carrythe pUC gene for ampicillin resistance and have HindIII, SphI, PstI andEcoRI sites available for the cloning of foreign sequences.

Example 22 Construction of Plant Expression Cassettes

[0529] Gene sequences intended for expression in transgenic plants arefirstly assembled in expression cassettes behind a suitable promoter andupstream of a suitable transcription terminator. These expressioncassettes can then be easily transferred to the plant transformationvectors described above in Example 21.

[0530] I. Promoter Selection

[0531] The selection of a promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and this selection will reflect the desiredlocation of expression of the transgene. Alternatively, the selectedpromoter may drive expression of the gene under a light-induced or othertemporally regulated promoter. A further alternative is that theselected promoter be chemically regulated. This would provide thepossibility of inducing expression of the transgene only when desiredand caused by treatment with a chemical inducer.

[0532] II. Transcriptional Terminators

[0533] A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tenterminator, the nopaline synthase terminator, the pea rbcS E9terminator, as well as terminators naturally associated with the plantprotox gene (i.e. “protox terminators”). These can be used in bothmonocotyledons and dicotyledons.

[0534] III. Sequences for the Enhancement or Regulation of Expression

[0535] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

[0536] Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize Adhl gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronzel gene had a similar effect in enhancing expression (Callis etal., supra). Intron sequences have been routinely incorporated intoplant transformation vectors, typically within the non-translatedleader.

[0537] A number of non-translated leader sequences derived from virusesare also known to enhance expression, and these are particularlyeffective in dicotyledonous cells. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic MottleVirus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res.15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79(1990))

[0538] IV. Targeting of the Gene Product Within the Cell

[0539] Various mechanisms for targeting gene products are known to existin plants and the sequences controlling the functioning of thesemechanisms have been characterized in some detail. For example, thetargeting of gene products to the chloroplast is controlled by a signalsequence that is found at the amino terminal end of various proteins andthat is cleaved during chloroplast import yielding the mature protein(e.g. Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signalsequences can be fused to heterologous gene products to effect theimport of heterologous products into the chloroplast (van den Broeck etal. Nature 313: 358-363 (1985)). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins that are known to be chloroplastlocalized.

[0540] Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting to cellular protein bodies has been described byRogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

[0541] In addition, sequences have been characterized that cause thetargeting of gene products to other cell compartments. Amino terminalsequences are responsible for targeting to the ER, the apoplast, andextracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, amino terminal sequences in conjunctionwith carboxy terminal sequences are responsible for vacuolar targetingof gene products (Shinshi et al., Plant Molec. Biol. 14: 357-368(1990)).

[0542] By the fusion of the appropriate targeting sequences describedabove to transgene sequences of interest it is possible to direct thetransgene product to any organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the amino terminal ATG of the transgene. The signal sequenceselected should include the known cleavage site and the fusionconstructed should take into account any amino acids after the cleavagesite that are required for cleavage. In some cases this requirement maybe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or alternatively replacement ofsome amino acids within the transgene sequence. Fusions constructed forchloroplast import can be tested for efficacy of chloroplast uptake byin vitro translation of in vitro transcribed constructions followed byin vitro chloroplast uptake using techniques described by (Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier. pp. 1081-1091 (1982); Wasmann et al. Mol. Gen. Genet. 205:446-453 (1986)). These construction techniques are well known in the artand are equally applicable to mitochondria and peroxisomes. The choiceof targeting that may be required for expression of the transgenes willdepend on the cellular localization of the precursor required as thestarting point for a given pathway. This will usually be cytosolic orchloroplastic, although it may is some cases be mitochondrial orperoxisomal. The products of transgene expression will not normallyrequire targeting to the ER, the apoplast or the vacuole.

[0543] The above described mechanisms for cellular targeting can beutilized not only in conjunction with their cognate promoters, but alsoin conjunction with heterologous promoters so as to effect a specificcell targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

Example 23 Transformation of Dicotyledons

[0544] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques that donot require Agrobacterium. Non-Agrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

[0545] Agrobacterium-mediated transformation is a preferred techniquefor transformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species. Themany crop species that are routinely transformable by Agrobacteriuminclude tobacco, tomato, sunflower, cotton, oilseed rape, potato,soybean, alfalfa and poplar (EP 0 317 511 (cotton), EP 0 249 432(tomato, to Calgene), WO 87/07299 (Brassica, to Calgene), U.S. Pat. No.4,795,855 (poplar)).

[0546] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols well known in the art.Transformed tissue is regenerated on selectable medium carrying theantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders.

Example 24 Transformation of Monocotyledons

[0547] Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complex vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

[0548] Patent Applications EP 0 292 435 (to Ciba-Geigy), EP 0 392 225(to Ciba-Geigy) and WO 93107278 (to Ciba-Geigy) describe techniques forthe preparation of callus and protoplasts from an elite inbred line ofmaize, transformation of protoplasts using PEG or electroporation, andthe regeneration of maize plants from transformed protoplasts.Gordon-Kamm et al., Plant Cell 2: 603-618 (1990)) and Fromm et al.,Biotechnology 8: 833-839 (1990)) have published techniques fortransformation of A188-derived maize line using particle bombardment.Furthermore, application WO 93/07278 (to Ciba-Geigy) and Koziel et al.,Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of elite inbred lines of maize by particle bombardment.This technique utilizes immature maize embryos of 1.5-2.5 mm lengthexcised from a maize ear 14-15 days after pollination and a PDS-1000HeBiolistics device for bombardment.

[0549] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).

[0550] Patent Application EP 0 332 581 (to Ciba-Geigy) describestechniques for the generation, transformation and regeneration ofPooideae protoplasts. These techniques allow the transformation ofDactylis and wheat. Furthermore, wheat transformation was been describedby Vasil et al., Biotechnology 10: 667-674 (1992)) using particlebombardment into cells of type C long-term regenerable callus, and alsoby Vasil et al., Biotechnology 11: 1553-1558 (1993)) and Weeks et al.,Plant Physiol. 102: 1077-1084 (1993) using particle bombardment ofimmature embryos and immature embryo-derived callus. A preferredtechnique for wheat transformation, however, involves the transformationof wheat by particle bombardment of immature embryos and includes eithera high sucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashige & Skoog, Physiologia Plantarum15:473497 (1962)) and 3 mg/1 2,4-D for induction of somatic embryos,which is allowed to proceed in the dark. On the chosen day ofbombardment, embryos are removed from the induction medium and placedonto the osmoticum (i.e. induction medium with sucrose or maltose addedat the desired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical. An appropriate gene-carryingplasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sizegold particles using standard procedures. Each plate of embryos is shotwith the DuPont Biolistics, helium device using a burst pressure of˜1000 psi using a standard 80 mesh screen. After bombardment, theembryos are placed back into the dark to recover for about 24 h (stillon osmoticum). After 24 hrs, the embryos are removed from the osmoticumand placed back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NAA, 5 mg/liter GA), further containing theappropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2mg/l methotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” that contained half-strength MS, 2% sucrose, and the sameconcentration of selection agent. Patent application WO 94/13822describes methods for wheat transformation and is hereby incorporated byreference.

Example 25 Isolation of the Arabidopsis thaliana Protox-1 PromoterSequence

[0551] A Lambda Zap II genomic DNA library prepared from Arabidopsisthaliana (Columbia, whole plant) was purchased from Stratagene.Approximately 125,000 phage were plated at a density of 25,000 pfu per15 cm Petri dish and duplicate lifts were made onto Colony/Plaque Screenmembranes (NEN Dupont). The plaque lifts were probed with theArabidopsis protox-l cDNA (SEQ ID NO:1 labeled with 32P-dCTP by therandom priming method (Life Technologies). Hybridization and washconditions were at 65° C. as described in Church and Gilbert, Proc.Natl. Acad. Sci. USA 81:1991-1995 (1984). Positively hybridizing plaqueswere purified and in vivo excised into pBluescript plasmids. Sequencefrom the genomic DNA inserts was determined by the chain terminationmethod using dideoxy terminators labeled with fluorescent dyes (AppliedBiosystems, Inc.). One clone, AraPT1Pro, was determined to contain580-bp of Arabidopsis sequence upstream from the initiating methionine(ATG) of the protox-1 protein coding sequence. This clone also containscoding sequence and introns that extend to-bp 1241 of the protox-1 cDNAsequence. The 580-bp 5′ noncoding fragment is the putative Arabidopsisprotox-1 promoter, and the sequence is set forth in SEQ ID NO:13.

[0552] AraPT1Pro was deposited Dec. 15, 1995, as pWDC-11 (NRRL #B-21515)

Example 26 Construction of Plant Transformation Vectors ExpressingAltered Protox-1 Genes Behind the Native Arabidopsis Protox-1 Promoter

[0553] A full-length cDNA of the appropriate altered Arabidopsisprotox-1 cDNA was isolated as an EcoRI-XhoI partial digest fragment andcloned into the plant expression vector pCGN1761ENX (see Example 9 ofInternational application no. PCT/IB95/00452 filed Jun. 8, 1995,published Dec. 21, 1995 as WO 95/34659). This plasmid was digested withNcoI and BamHI to produce a fragment comprised of the complete protox-1cDNA plus a transcription terminator from the 3′ untranslated sequenceof the tml gene of Agrobacterium tumefaciens. The AraPT1Pro plasmiddescribed above was digested with NcoI and BamHI to produce a fragmentcomprised of pBluescript and the 580-bp putative Arabidopsis protox-1promoter. Ligation of these two fragments produced a fusion of thealtered protox cDNA to the native protox promoter. The expressioncassette containing the protox-1 promoter/protox-1 cDNA/tml terminatorfusion was excised by digestion with KpnI and cloned into the binaryvector pCIB200. The binary plasmid was transformed by electroporationinto Agrobacterium and then into Arabidopsis using the vacuuminfiltration method (Bechtold et al., CR. Acad. Sci. Paris 316:1194-1199 (1993). Transformants expressing altered protox genes wereselected on kanamycin or on various concentrations of protox inhibitingherbicide.

Example 27 Production of Herbicide Tolerant Plants by Expression of aNative Protox-1 Promoter/Altered Protox-1 Fusion

[0554] Using the procedure described above, an Arabidopsis protox-1 cDNAcontaining a TAC to ATG (Tyrosine to Methionine) change at nucleotides1306-1308 in the protox-1 sequence (SEQ ID NO:1) was fused to the nativeprotox-1 promoter fragment and transformed into Arabidopsis thaliana.This altered protox-1 enzyme (AraC-2Met) has been shown to be >10-foldmore tolerant to various protox-inhibiting herbicides than the naturallyoccurring enzyme when tested in the previously described bacterialexpression system. Seed from the vacuum infiltrated plants was collectedand plated on a range (10.0 nM-1.0 uM) of a protox inhibitory aryluracilherbicide of formula XVII. Multiple experiments with wild typeArabidopsis have shown that a 10.0 nM concentration of this compound issufficient to prevent normal seedling germination. Transgenic seedsexpressing the AraC-2Met altered enzyme fused to the native protox-1promoter produced normal Arabidopsis seedlings at herbicideconcentrations up to 500 nM, indicating at least 50-fold higherherbicide tolerance when compared to wild-type Arabidopsis. Thispromoter/altered protox enzyme fusion therefore functions as aneffective selectable marker for plant transformation. Several of theplants that germinated on 100.0 nM of protox-inhibiting herbicide weretransplanted to soil, grown 2-3 weeks, and tested in a spray assay withvarious concentrations of the protox-inhibiting herbicide. When comparedto empty vector control transformants, the AraPT1Pro/AraC-2Mettransgenics were >10-fold more tolerant to the herbicide spray.

Example 28 Demonstration of resistant mutations' cross-tolerance tovarious protox-inhibiting compounds in an Arabidopsis germination assay

[0555] Using the procedure described above, an Arabidopsis protox-1 cDNAcontaining both a TAC to ATC (tyrosine to isoleucine) change atnucleotides 1306-1308 and a TCA to TTA (serine to leucine) change atnucleotides 945-947 in the protox-1 sequence (SEQ ID NO:1) was fused tothe native protox-1 promoter fragment and transformed into Arabidopsisthaliana. This altered protox-1 enzyme (AraC-2Ile+AraC305Leu) has beenshown to be >10-fold more tolerant to a protox inhibitory aryluracilherbicide of formula XVII than the naturally occurring enzyme whentested in a bacterial system (see Examples 9-13). Homozygous Arabidopsislines containing this fusion were generated from transformants thatshowed high tolerance to a protox inhibiting herbicide in a seedlinggermination assay as described above. The seed from one line was testedfor cross-tolerance to various protox-inhibitory compounds by repeatingthe germination assay on concentrations of the compounds that had beenshown to inhibit germination of wild-type Arabidopsis. The results fromthese experiments are shown in Table 4.

Example 29 Isolation of a Maize Protox-1 Promoter Sequence

[0556] A Zea Mays (Missouri 17 inbred, etiolated seedlings) genomic DNAlibrary in the Lambda FIX II vector was purchased from Stratagene.Approximately 250,000 pfu of the library was plated at a density of50,000 phage per 15 cm plate and duplicate lifts were made ontoColony/Plaque screen membranes (NEN Dupont). The plaque lifts wereprobed with the maize protox-1 cDNA (SEQ ID NO:5) labeled with 32P-dCTPby the random priming method (Life Technologies). Hybridization and washconditions were at 65° C. as described in Church and Gilbert, Proc.Natl. Acad. Sci. USA 81: 1991-1995 (1984). Lambda phage DNA was isolatedfrom three positively hybridizing phage using the Wizard Lambda PrepsDNA Purification System (Promega). Analysis by restriction digest,hybridization patterns, and DNA sequence analysis identified a lambdaclone containing approximately 3.5 kb of maize genomic DNA located 5′ tothe maize protox-1 coding sequence previously isolated as a cDNA clone.This fragment includes the maize protox-1 promoter. The sequence of thisfragment is set forth in SEQ ID NO:14. From nucleotide 1 to 3532, thissequence is comprised of 5′ noncoding sequence. From nucleotide 3533 to3848, this sequence encodes the 5′ end of the maize protox-1 protein.

[0557] A plasmid containing the sequence of SEQ ID NO:14 fused to theremainder of the maize protox-1 coding sequence was deposited Mar. 19,1996 as pWDC-14 (NRRL #B-21546).

Example 30 Construction of Plant Transformation Vectors ExpressingAltered Protox-1 Genes Behind the Native Maize Protox-1 Promoter

[0558] The 3848-bp maize genomic fragment (SEQ ID NO:14) was excisedfrom the isolated lambda phage clone as a SalI-KpnI partial digestproduct and ligated to a KpnI-NotI fragment derived from an alteredmaize protox-1 cDNA that contained an alanine to leucine change at aminoacid 164 (SEQ ID NO:6). This created a fusion of the native maizeprotox-1 promoter to a full length cDNA that had been shown to conferherbicide tolerance in a bacterial system (Examples 9-14). This fusionwas cloned into a pUC18 derived vector containing the CaMV 35Sterminator sequence to create a protox promoter/altered protoxcDNA/terminator cassette. The plasmid containing this cassette wasdesignated pWCo-1.

[0559] A second construct for maize transformation was created byengineering the first intron found in the coding sequence from the maizegenomic clone back into the maize cDNA. The insertion was made usingstandard overlapping PCR fusion techniques. The intron (SEQ ID NO:25)was 93-bp long and was inserted between nucleotides 203 and 204 of SEQID NO:6, exactly as it appeared in natural context in the lambda clonedescribed in Example 29. This intron-containing version of theexpression cassette was designated pWCo-2.

Example 31 Demonstration of Maize Protox-1 Promoter Activity inTransgenic Maize Plants

[0560] Maize plants transformed with maize protox promoter/alteredprotox fusions were identified using PCR analysis with primers specificfor the transgene. Total RNA was prepared from the PCR positive plantsand reverse-transcribed using Superscript M-MLV (Life Technologies)under recommended conditions. Two microliters of the reversetranscription reaction was used in a PCR reaction designed to bespecific for the altered protox sequence. While untransformed controlsgive no product in this reaction, approximately 85% of plantstransformed with pWCo-1 gave a positive result, indicating the presenceof mRNA derived from the transgene. This demonstrates some level ofactivity for the maize protox promoter. The RNA's from the transgenicmaize plants were also subjected to standard northern blot analysisusing the radiolabeled maize protox cDNA fragment from SEQ ID NO:6 as aprobe. Protox-1 mRNA levels significantly above those of untransformedcontrols were detected in some of the transgenic maize plants. Thiselevated mRNA level is presumed to be due to expression of alteredprotox-1 mRNA from the cloned maize protox promoter.

Example 32 Isolation of a Sugar Beet Protox-1 Promoter Sequence

[0561] A genomic sugar beet library was prepared by Stratagene in theLambda Fix II vector. Approximately 300,000 pfu of the library wasplated and probed with the sugar beet protox-1 cDNA sequence (SEQ IDNO:17) as described for maize in Example 29. Analysis by restrictiondigest, hybridization patterns and DNA sequence analysis identified alambda clone containing approximately 7 kb of sugar beet genomic DNAlocated 5′ to the sugar beet coding sequence previously isolated as acDNA clone. A PstI-SalI fragment of 2606-bp was subcloned from thelambda clone into a pBluescript vector. This fragment contains 2068-bpof 5′ noncoding sequence and includes the sugar beet protox-1 promotersequence. It also includes the first 453-bp of the protox-1 codingsequence and the 85-bp first intron contained in the coding sequence.The sequence of this fragment is set forth in SEQ ID NO:26.

[0562] A plasmid containing the sequence of SEQ ID NO:26 was depositedDec. 6, 1996 as pWDC-20 (NRRL #B-21650).

Example 33 Construction of Plant Transformation Vectors ExpressingAltered Sugar Beet Protox-1 Genes Behind the Native Sugar Beet Protox-1Promoter

[0563] The sugar beet genomic fragment (SEQ ID NO:26) was excised fromthe genomic subclone described in Example 32 as a SacI-BsrGI fragmentthat includes 2068-bp of 5′ noncoding sequence and the first 300-bp ofthe sugar beet protox-1 coding sequence. This fragment was ligated to aBsrGI-NotI fragment derived from an altered sugar beet protox-1 cDNAthat contained a tyrosine to methionine change at amino acid 449 (SEQ IDNO:18). This created a fusion of the native sugar beet protox-1 promoterto a full length cDNA that had been shown to confer herbicide tolerancein a bacterial system (Examples 9-14). This fusion was cloned into apUC18 derived vector containing the CaMV 35S terminator sequence tocreate a protox promoter/altered protox cDNA/terminator cassette. Theplasmid containing this cassette was designated pWCo-3.

Example 34 Production of Herbicide Tolerant Plants by Expression of aNative Sugar Beet Protox-1 Promoter/Altered Sugar Beet Protox-1 Fusion

[0564] The expression cassette from pWCo-3 is transformed into sugarbeet using any of the transformation methods applicable to dicot plants,including Agrobacterium, protoplast, and biolistic transformationtechniques. Transgenic sugar beets-expressing the altered protox-1enzyme are identified by RNA-PCR and tested for tolerance toprotox-inhibiting herbicides at concentrations that are lethal tountransformed sugar beets.

Section D: Expression of Protox Genes in Plant Plastids Example 35Preparation of a Chimeric Gene Containing the Tobacco Plastid clpP GenePromoter and Native clpP 5′ Untranslated Sequence Fused to a GUSReporter Gene and Plastid rps16 Gene 3′ Untranslated Sequence in aPlastid Transformation Vector

[0565] I. Amplification of the Tobacco Plastid clpP Gene Promoter andComplete 5′ Untranslated RNA (5′ UTR).

[0566] Total DNA from N. tabacum c.v. “Xanthi NC” was used as thetemplate for PCR with a left-to-right “top strand” primer comprising anintroduced EcoRI restriction site at position -197 relative to the ATGstart codon of the constitutively expressed plastid clpP gene (primerPclp_P1a: 5′-GCG+E,uns GAATTCATACTTATTTATCATTAGAAAG-3′ (SEQ ID NO:27);EcoRI restriction site underlined) and a right-to-left “bottom strand”primer homologous to the region from −21 to −1 relative to the ATG startcodon of the clpP promoter that incorporates an introduced NcoIrestriction site at the start of translation (primer Pclp_P2b:5′-GCG+E,uns CCATGGTAAATGAAAGAAAGAACTAAA-3′ (SEQ ID NO:28); NcoIrestriction site underlined). This PCR reaction was undertaken with Pfuthermostable DNA polymerase (Stratagene, La Jolla Calif.) in a PerkinElmer Thermal Cycler 480 according to the manufacturer's recommendations(Perkin Elmer/Roche, Branchburg, N.J.) as follows: 7 min 95° C.,followed by 4 cycles of 1 min 95° C./2 min 43° C./1 min 72° C., then 25cycles of 1 min 95° C./2 min 55° C./1 min 72° C. The 213-bpamplification product comprising the promoter and 5′ untranslated regionof the clpP gene containing an EcoRI site at its left end and an NcoIsite at its right end and corresponding to nucleotides 74700 to 74505 ofthe N. tabacum plastid DNA sequence (Shinozaki et al., EMBO J. 5:2043-2049 (1986)) was gel purified using standard procedures anddigested with EcoRI and NcoI (all restriction enzymes were purchasedfrom New England Biolabs, Beverly, Mass.).

[0567] II. Amplification of the Tobacco Plastid rps16 Gene 3′Untranslated RNA Sequence (3′UTR).

[0568] Total DNA from N. tabacum c.v. “Xanthi NC” was used as thetemplate for PCR as described above with a left-to-right “top strand”primer comprising an introduced XbaI restriction site immediatelyfollowing the TAA stop codon of the plastid rps16 gene encodingribosomal protein S16 (primer rps16P_(—)1a (5′-GCG+E,unsTCTAGATCAACCGAAATTCAATTAAGG-3′ (SEQ ID NO:30); XbaI restriction siteunderlined) and a right-to-left “bottom strand” primer homologous to theregion from +134 to +151 relative to the TAA stop codon of rps16 thatincorporates an introduced HindIII restriction site at the 3′ end of therps16 3′ UTR (primer rps16P_(—)1b (5′-CGC+E,unsAAGCTTCAATGGAAGCAATGATAA-3′ (SEQ ID NO:31); HindIII restriction siteunderlined). The 169-bp amplification product comprising the 3′untranslated region of the rps16 gene containing an XbaI site at itsleft end and a HindIII site at its right end and containing the regioncorresponding to nucleotides 4943 to 5093 of the N. tabacum plastid DNAsequence (Shinozaki et al., 1986) was gel purified and digested withXbaI and HindIII.

[0569] III. Ligation of a GUS Reporter Gene Fragment to the clpP GenePromoter and 5′ and 3′ UTR's.

[0570] An 1864-bp β-glucuronidase (GUS) reporter gene fragment derivedfrom plasmid pRAJ275 (Clontech) containing an NcoI restriction site atthe ATG start codon and an XbaI site following the native 3′ UTR wasproduced by digestion with NcoI and XbaI. This fragment was ligated in afour-way reaction to the 201-bp EcoRI/NcoI clpP promoter fragment, the157-bp XbaI/HindIII rps16 3′UTR fragment, and a 3148-bp EcoRI/HindIIIfragment from cloning vector pGEM3Zf(−) (Promega, Madison Wis.) toconstruct plasmid pPH138. Plastid transformation vector pPH140 wasconstructed by digesting plasmid pPRV111a (Zoubenko et al. 1994) withEcoRI and HindIII and ligating the resulting 7287-bp fragment to a2222-bp EcoRI/HindIII fragment of pPH138.

Example 36 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter Plus Tobacco Plastid psbA Gene Minimal 5′Untranslated Sequence Fused to a GUS Reporter Gene and Plastid rps16Gene 3′ Untranslated Sequence in a Plastid Transformation Vector

[0571] Amplification of the tobacco plastid clpP gene promoter andtruncated 5′ untranslated RNA (5′ UTR): Total DNA from N. tabacum c.v.“Xanthi NC” was used as the template for PCR as described above with theleft-to-right “top strand” primer Pclp_Pla (SEQ ID NO:27) and aright-to-left “bottom strand” primer homologous to the region from −34to −11 relative to the ATG start codon of the clpP promoter thatincorporates an introduced XbaI restriction site in the clpP 5′ UTR atposition −11 (primer Pclp_P1b: 5′-GCG+E,unsTCTAGAAAGAACTAAATACTATATTTCAC-3′ (SEQ ID NO:29); XbaI restriction siteunderlined). The 202-bp amplification product comprising the promoterand truncated 5′ UTR of the clpP gene containing an EcoRI site at itsleft end and an XbaI site at its right end was gel purified and digestedwith XbaI. The XbaI site was subsequently filled in with Klenow DNApolymerase (New England Biolabs) and the fragment digested with EcoRI.This was ligated in a five-way reaction to a double stranded DNAfragment corresponding to the final 38 nucleotides and ATG start codonof the tobacco plastid psbA gene 5′ UTR (with an NcoI restriction siteoverhang introduced into the ATG start codon) that was created byannealing the synthetic oligonucleotides minpsb_U (top strand:5′-GGGAGTCCCTGATGATTAAATAAACCAAGATTTTAC-3′ (SEQ ID NO:32)) and minpsb_L(bottom strand: 5′-+E,uns CATGGTAAAATCTTGGTTTATTTAATCATCAGGGACTCCC-3′(SEQ ID NO:33); NcoI restriction site 5′ overhang underlined), theNcoI/XbaI GUS reporter gene fragment described above, the XbaI/HindIIIrps16 3′ UTR fragment described above, and the EcoRI/HindIII pGEM3Zf(−)fragment described above to construct plasmid pPH139. Plastidtransformation vector pPH144 was constructed by digesting plasmidpPRV111a (Zoubenko, et al., Nucleic Acids Res 22: 3819-3824 (1994)) withEcoRI and HindIII and ligating the resulting 7287-bp fragment to a2251-bp EcoRI/HindIII fragment of pPH139.

Example 37 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter and Complete 5′ Untranslated Sequence Fused to theArabidopsis thaliana Protox-1 Coding Sequence and Plastid rps16 Gene 3′Untranslated Sequence in a Vector for Tobacco Plastid Transformation

[0572] Miniprep DNA from plasmid AraC-2Met carrying an Arabidopsisthaliana NotI insert that includes cDNA sequences from theProtoporphyrinogen IX Oxidase (“protox”) gene encoding a portion of theamino terminal plastid transit peptide, the full-length cDNA and aportion of the 3′ untranslated region was used as the template for PCRas described above using a left-to-right “top strand” primer (withhomology to nucleotides +172 to +194 relative to the ATG start codon ofthe full length precursor protein) comprising an introduced NcoIrestriction site and new ATG start codon at the deduced start of themature protox protein coding sequence (primer APRTXP1a: 5′-GGGA+E,unsCCATGGATTGTGTGATTGTCGGCGGAGG-3′ (SEQ ID NO:34); NcoI restriction siteunderlined) and a right-to-left “bottom strand” primer homologous tonucleotides +917 to +940 relative to the native ATG start codon of theprotox precursor protein (primer APRTXP1b: 5′-CTCCGCTCTCCAGCTTAGTGATAC-3′ (SEQ ID NO:35)). The 778-bp product wasdigested with NcoI and SfuI and the resulting 682-bp fragment ligated toan 844-bp SfuI/NotI DNA fragment of AraC-2Met comprising the 3′ portionof the protox coding sequence and a 2978-bp NcoI/NotI fragment of thecloning vector pGEM5Zf(+) (Promega, Madison Wis.) to construct plasmidpPH141. Plastid transformation vector pPH143 containing the clpPpromoter driving the Formula XVII-resistant AraC-2Met protox gene withthe rps16 3′ UTR was constructed by digesting pPH141 with NcoI and SspIand isolating the 1491-bp fragment containing the complete protox codingsequence, digesting the rps16P_(—)1a and rps16P_(—)1b PCR productdescribed above with HindIII, and ligating these to a 7436-bpNcoI/HindIII fragment of pPH140.

Example 38 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter Plus Tobacco Plastid psbA Gene Minimal 5′Untranslated Sequence Fused to the Arabidopsis thaliana Protox-1 CodingSequence and Plastid rps16 Gene 3′ Untranslated Sequence in a Vector forTobacco Plastid Transformation

[0573] Plastid transformation vector pPH145 containing the clpPpromoter/psbA 5′ UTR fusion driving the Formula XVII-resistant AraC-2Metprotox gene with the rps16 3′ UTR was constructed by digesting pPH141with NcoI and SspI and isolating the 1491-bp fragment containing thecomplete protox coding sequence, digesting the rps16P_(—)1a andrps16P_(—)1b PCR product described above with HindIII, and ligatingthese to a 7465-bp NcoI/HindIII fragment of pPH144.

Example 39 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter and 5′ Untranslated Sequence Fused to the EPSPSynthase Coding Sequence and Plastid rps16 Gene 3′ Untranslated Sequencein a Vector for Tobacco Plastid Transformation

[0574] A cDNA library is screened for the5-enolpyruvyl-3-phosphoshikimate synthase (EPSP synthase) gene (U.S.Pat. Nos. 5,310,667, 5,312,910, and 5,633,435, all incorporated hereinby reference). A plasmid clone containing the full length EPSP synthasegene cDNA is isolated by standard techniques of molecular cloning. PCRprimers are designed for amplification of the mature-size EPSP synthasecoding sequence from this plasmid using a top strand primer having a 5′extension containing an NcoI restriction site inserted at amino acid -1from the deduced mature protein start, thus creating an ATG start codonat this position, and a bottom strand primer having a 5′ extensioncontaining an XbaI restriction site downstream of the stop codon of theEPSP mature coding sequence in the amplified PCR product. The PCRamplification is performed using the designated primers and plasmid DNAtemplate according to standard protocols. Amplified products are clonedand sequenced and a NcoI-XbaI DNA fragment containing the completemature EPSP synthase coding sequence is isolated by restriction digestwith NcoI and XbaI, electrophoresis on a 0.8% TAE agarose gel, andphenol extraction of the excised band.

[0575] A plastid transformation vector containing the clpP promoterdirecting transcription of the mature-sized EPSP synthase gene with therps16 3′ UTR is constructed by digesting pPH140 with NcoI and XbaI andpurifying the fragment containing the vector backbone, 5′ and 3′ plastidintegration targeting sequences, aada selectable marker cassette, andclpP promoter/rps16 3′ UTR expression sequences. This product is ligatedin a two-way reaction with the NcoI-XbaI DNA fragment containing themature-sized EPSP synthase coding sequence isolated as described above.

Example 40 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter and 5′ Untranslated Sequence Fused to the ALS CodingSequence and Plastid rps16 Gene 3′ Untranslated Sequence in a Vector forTobacco Plastid Transformation

[0576] A cDNA library is screened for the acetolactate synthase (ALS)gene (U.S. Pat. No. 5,013,659). A plasmid clone containing the fulllength ALS gene cDNA is isolated by standard techniques of molecularcloning. PCR primers are designed for amplification of the mature-sizeALS coding sequence from this plasmid using a top strand primer having a5′ extension containing an NcoI restriction site inserted at amino acid-1 from the deduced mature protein start, thus creating an ATG startcodon at this position, and a bottom strand primer having a 5′ extensioncontaining an XbaI restriction site downstream of the stop codon of theALS mature coding sequence in the amplified PCR product. The PCRamplification is performed using the designated primers and plasmid DNAtemplate according to standard protocols. Amplified products are clonedand sequenced and a NcoI-XbaI DNA fragment containing the completemature ALS coding sequence is isolated by restriction digest with NcoIand Xbal, electrophoresis on a 0.8% TAE agarose gel, and phenolextraction of the excised band.

[0577] A plastid transformation vector containing the clpP promoterdriving the mature-sized ALS gene with the rps16 3′ UTR is constructedby digesting pPH140 with NcoI and Xbal and purifying the fragmentcontaining the vector backbone, 5′ and 3′ plastid integration targetingsequences, aada selectable marker cassette, and clpP promoter/rps16 3′UTR expression sequences. This product is ligated in a two-way reactionwith the NcoI-XbaI DNA fragment containing the mature-sized ALS codingsequence isolated as described above.

Example 41 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene Promoter and 5′ Untranslated Sequence Fused to the AHAS CodingSequence and Plastid rps16 Gene 3′ Untranslated Sequence in a Vector forTobacco Plastid Transformation

[0578] A cDNA library is screened for the acetohydroxyacid synthase(AHAS) gene (U.S. Pat. No. 4,761,373). A plasmid clone containing thefull length AHAS gene cDNA is isolated by standard techniques ofmolecular cloning. PCR primers are designed for amplification of themature-size AHAS coding sequence from this plasmid using a top strandprimer having a 5′ extension containing an NcoI restriction siteinserted at amino acid −1 from the deduced mature protein start, thuscreating an ATG start codon at this position, and a bottom strand primerhaving a 5′ extension containing an XbaI restriction site downstream ofthe stop codon of the AHAS mature coding sequence in the amplified PCRproduct. The PCR amplification is performed using the designated primersand plasmid DNA template according to standard protocols. Amplifiedproducts are cloned and sequenced and a NcoI-XbaI DNA fragmentcontaining the complete mature AHAS coding sequence is isolated byrestriction digest with NcoI and XbaI, electrophoresis on a 0.8% TAEagarose gel, and phenol extraction of the excised band.

[0579] A plastid transformation vector containing the clpP promoterdriving the mature-sized AHAS gene with the rps16 3′ UTR is constructedby digesting pPH140 with NcoI and XbaI and purifying the fragmentcontaining the vector backbone, 5′ and 3′ plastid integration targetingsequences, aada selectable marker cassette, and clpP promoter/rps16 3′UTR expression sequences. This product is ligated in a two-way reactionwith the NcoI-XbaI DNA fragment containing the mature-sized AHAS codingsequence isolated as described above.

Example 42 Preparation of a Chimeric Gene Containing the Tobacco PlastidclpP Gene

[0580] Promoter and 5′ Untranslated Sequence Fused to the ACCase CodingSequence and Plastid rps16 Gene 3′ Untranslated Sequence in a Vector forTobacco Plastid Transformation

[0581] A cDNA library is screened for the acetylcoenzyme A carboxylase(ACCase) gene (U.S. Pat. No. 5,162,602). A plasmid clone containing thefull length ACCase gene cDNA is isolated by standard techniques ofmolecular cloning. PCR primers are designed for amplification of themature-size ACCase coding sequence from this plasmid using a top strandprimer having a 5′ extension containing an NcoI restriction siteinserted at amino acid -1 from the deduced mature protein start, thuscreating an ATG start codon at this position, and a bottom strand primerhaving a 5′ extension containing an XbaI restriction site downstream ofthe stop codon of the ACCase mature coding sequence in the amplified PCRproduct. The PCR amplification is performed using the designated primersand plasmid DNA template according to standard protocols. Amplifiedproducts are cloned and sequenced and a NcoI-XbaI DNA fragmentcontaining the complete mature ACCase coding sequence is isolated byrestriction digest with NcoI and XbaI, electrophoresis on a 0.8% TAEagarose gel, and phenol extraction of the excised band.

[0582] A plastid transformation vector containing the clpP promoterdriving the mature-sized ACCase gene with the rps16 3′ UTR isconstructed by digesting pPH140 with NcoI and XbaI and purifying thefragment containing the vector backbone, 5′ and 3′ plastid integrationtargeting sequences, aada selectable marker cassette, and clpPpromoter/rps16 3′ UTR expression sequences. This product is ligated in atwo-way reaction with the NcoI-XbaI DNA fragment containing themature-sized ACCase coding sequence isolated as described above.

Example 43 Biolistic Transformation of the Tobacco Plastid Genome

[0583] Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ were germinated sevenper plate in a 1″ circular array on T agar medium and bombarded 12-14days after sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 andpPH145 essentially asdescribed in Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917. Bombardedseedlings were incubated on T medium for two days after which leaveswere excised and placed abaxial side up in bright light (350-500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentwere subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones was assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) was separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

Example 44 Assessment of Herbicide Tolerance in Nt_pPH143 and Nt_pPH145Plastid Transformant Lines

[0584] Primary homoplasmic transformant lines transformed with pPH143(line Nt_pPH143) or with pPH145 (line Nt_pPH145), which were obtained asdescribed in Example 43, were grown to maturity in the greenhouse.Flowers were either: (a) self-pollinated, (b) pollinated with wildtypetobacco (c.v. Xanthi nc), or (c) used as pollen donors to fertilizeemasculated flowers of wildtype Xanthi plants. Plastid segregation ofthe linked spectinomycin resistance marker was verified by uniparentalfemale inheritance of the spectinomycin-resistance phenotype in eachtransformant line using a minimum of 50 seeds per selection pool derivedfrom either selfed or backcross capsules. Additional self or wildtypebackcross (Xanthi pollen parent) seeds were germinated in soil. 36plants of each line (143 1B-1, 143 1B-4, 143 4A-2, 143 4A-5; 145 7A-5,145 7A-6, 145 8A-3) plus 36 wildtype Xanthi plants as isogenic controlswere grown in separate 6″ clay pots in a controlled environment cubicle.In order to assess tolerance to the protox inhibitor Formula XVII,plants of Xanthi and the seven transformant lines were distributed intoeight identical 16-pot flats (2 plants of each type per flat). The flatswere sprayed with Formula XVII until runoff at concentrations of either0, 0.5, 2.5, 5, 10, 25, 50, or 100 mg Formula XVII per liter. Solutionswere made up in water using 4 g/liter or 40 g/liter stock solutions ofFormula XVII dissolved in dimethylsulfoxide (DMSO) and used immediatelyafter preparation. Twenty microliters of the wetting agent Silwett wasadded to each 200 ml volume of herbicide solution for a finalconcentration of 0.01%. Flats were sprayed in the late afternoon andallowed to dry overnight before transfer to the growth cubicle.Tolerance was assesed by comparing leaf damage and wilting to theuntransformed Xanthi controls at 0, 18 hrs, 48 hrs, and 6 dayspost-application. Severe damage was apparent on the Xanthi plants at allconcentrations above 0.5 mg/l, and complete wilting/burn down occurredabove 2.5 mg/l. Only slight damage occurred on the Nt_pPH143 plants evenat the highest concentration (100 mg/liter), and the plants soon outgrewthe bleached spots (the appearance of Xanthi at 0.5 mg/liter wasapproximately equivalent to Nt_pPH143 1 B-1 at 100 mg/liter, giving atolerance of ca. 200-fold).

Example 45 Plastid Transformation of Maize

[0585] Type I embryogenic callus cultures (Green et al. (1983) in A.Fazelahmad, K. Downey, J. Schultz, R. W. Voellmy, eds. Advances in GeneTechnology: Molecular Genetics of Plants and Animals. Miami WinterSymposium Series, Vol. 20. Academic Press, N.Y.) of the proprietarygenotypes CG00526 and CG00714 are initiated from immature embryos, 1.5-2.5 mm in length, from greenhouse grown material. Embryos areaseptically excised from surface-sterilized ears approximately 14 daysafter pollination. Embryos of CG00526 are placed on D callus initiationmedia with 2% sucrose and 5mg/L chloramben (Duncan et al. (1985) Planta165: 322-332) while those of CG00714 are placed onto KM callusinitiation media with 3% sucrose and 0.75mg/L 2,4-d (Kao and Michayluk(1975) Planta 126, 105-110). Embryos and embryogenic cultures aresubsequently cultured in the dark. Embryogenic responses are removedfrom the explants after ˜14 days. CGO0526 responses are placed onto Dcallus maintenance media with 2% sucrose and 0.5mg/L 2,4-d while thoseof CG00714 are placed onto KM callus maintenance media with 2% sucroseand 5mg/L Dicamba. After 3 to 8 weeks of weekly selective subculture tofresh maintenance media, high quality compact embryogenic cultures areestablished. Actively growing embryogenic callus pieces are selected astarget tissue for gene delivery. The callus pieces are plated ontotarget plates containing maintenance medium with 12% sucroseapproximately 4 hours prior to gene delivery. The callus pieces arearranged in circles, with radii of 8 and 10 mm from the center of thetarget plate. Plasmid DNA is precipitated onto gold microcarriers asdescribed in the DuPont Biolistics manual. Two to three μg of eachplasmid is used in each 6 shot microcarrier preparation. Genes aredelivered to the target tissue cells using the PDS-1000He Biolisticsdevice. The settings on the Biolistics device are as follows: 8 mmbetween the rupture disc and the macrocarrier, 10 mm between themacrocarrier and the stopping screen and 7 cm between the stoppingscreen and the target. Each target plate is shot twice using 650 psirupture discs. A 200×200 stainless steel mesh (McMaster-Carr, NewBrunswick, N.J.) is placed between the stopping screen and the targettissue.

[0586] Five days later, the bombed callus pieces are transferred tomaintenance medium with 2% sucrose and 0.5mg/L 2,4-d, but without aminoacids, and containing 750 or 1000 nM Formula XVII. The callus pieces areplaced for 1 hour on the light shelf 4-5 hours after transfer or on thenext day, and stored in the dark at 27° C. for 5-6 weeks. Following the5-6 week primary selection stage, yellow to white tissue is transferredto fresh plates containing the same medium supplemented with 500 or 750nM Formula XVII. 4-5 hours after transfer or on the next day, thetissues are placed for 1 hour on the light shelf and stored in the darkat 27° C. for 3-4 weeks. Following the 3-4 week secondary selectionstage, the tissues are transferred to plates containing the same mediumsupplemented with 500 nM Formula XVII. Healthy growing tissue is placedfor 1 hour on the light shelf and stored in the dark at 27° C. It issubcultured every two weeks until the colonies are large enough forregeneration.

[0587] At that point, colonies are transferred to a modified MS medium(Murashige and Skoog (1962) Physiol. Plant 15: 473-497) containing 3%sucrose (MS3S) with no selection agent and placed in the light. ForCG00526, 0.25mg/L ancymidol and 0.5mg/L kinetin are added to this mediumto induce embryo germination, while for CG00714, 2mg/L benzyl adenine isadded. Regenerating colonies are transferred to MS3S media withoutancymidol and kinetin, or benzyl adenine, for CG00526 or CG00714,respectively, after 2 weeks. Regenerating shoots with or without rootsare transferred to boxes containing MS3S medium and small plants withroots are eventually recovered and transferred to soil in thegreenhouse.

Section E. Herbicide Tolerant Protox Genes Produced By DNA ShufflingExample 46 In vitro Recombination of Protox Genes by DNA Shuffling

[0588] One of the plant protox genes described herein (SEQ ID NO:1, 3,5, 7, 9, 11, 15, 17, 19, 21, 23, or 36) or one of the above-describedinhibitor-resistant mutants thereof is amplified by PCR. The resultingDNA fragment is digested by DNaseI treatment essentially as described inStemmer et al., PNAS 91: 10747-10751 (1994), and the PCR primers areremoved from the reaction mixture. A PCR reaction is carried out withoutprimers and is followed by a PCR reaction with the primers, both asdescribed in Stemmer et al. (1994). The resulting DNA fragments arecloned into pTRC99a (Pharmacia, Cat no: 27-5007-01) and transformed intoE. coli strain SASX38 by electroporation using the Biorad Gene Pulserand the manufacturer's conditions. The transformed bacteria are grown onmedium that contains inhibitory concentrations of the inhibitor andthose colonies that grow in the presence of the inhibitor are selected.Colonies that grow in the presence of normally inhibitory concentrationsof inhibitor are picked and purified by repeated restreaking. Theirplasmids are purified and the DNA sequences of cDNA inserts fromplasmids that pass this test are then determined.

[0589] In a similar reaction, PCR-amplified DNA fragments comprising oneof the plant protox genes described herein (SEQ ID NO:1, 3, 5, 7, 9, 11,15, 17, 19, 21, 23, or 36, or an inhibitor-resistant mutants thereof),and PCR-amplified DNA fragments comprising at least one other of theplant protox genes described herein (or an inhibitor-resistant mutantthereof) are recombined in vitro and resulting variants with improvedtolerance to the inhibitor are recovered as described above.

Example 47 In vitro Recombination of Protox Genes by Staggered ExtensionProcess

[0590] Two or more of the plant protox genes described herein (selectedfrom SEQ ID NO:1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, and 36, or aninhibitor-resistant mutant thereof) are each cloned into the polylinkerof a pBluescript vector. A PCR reaction is carried out essentially asdescribed in Zhao et al., Nature Biotechnology 16: 258-261 (1998) usingthe “reverse primer” and the “M13 20 primer” (Stratagene Catalog).Amplified PCR fragments are digested with appropriate restrictionenzymes and cloned into pTRC99a and mutated protox genes with improvedtolerance to the inhibitor are recovered as described above. TABLE 1AAlignment of the full-length and partial protox-1 amino acid sequencesfrom Arabidopsis (“Arabpt-1”; SEQ ID NO:2), maize (“Mzpt-1”; SEQ IDNO:6), wheat (“Wtpt-1”; SEQ ID NO:10), soybean (“Soybeanpt-1”; SEQ IDNO:12), cooton (“Cottonpt-1”; SEQ ID NO:16), sugar beet (“Sugpt-1”; SEQID NO:18), oilseed rape (“Rapept-1”; SEQ ID NO:20), rice (“Ricept-1”;SEQ ID NO:22), sorghum (“Sorghumpt-1”; SEQ ID NO:24), and sugar cane(“Scpt-1”; SEQ ID NO:37). Alignment was performed using the PileUpprogram (GCG package, University of Wisconsin, Madison, WI). Positionsthat may be modified according to the teachings herein to confer orenhance inhibitor resistance are shown in bold type. 1 50 Rapept-1.......... .......... MDLSLLRP.. QPFLSPFSNP FPRSRPYKPL Arabpt-1.......... .......... MELSLLRPTT QSLLPSFSKP NLRLNVYKPL Sorghumpt-1.......... .......... .......... .......... .......... Mzpt-1 .................... .......... .......... .......... Wtpt-1 .......... ...................M ATATVAAASP LRGRVTGRPH Ricept-1 .......... .................... .......... .......... Cottonpt-1 .......... ......MTALIDLSLLRSSP SVSPFSIPHH RKFPRSRPNP Sugpt-1 MKSMALSNCI PQTQCMPLRSSGHYRGNCIM LSIPCSLIGR RGYYSHKKRR Scpt-1 .......... .......... .................... .......... 51 100 Rapept-1 NLRCSVSGGS VVGSSTIEGG GGGKTVTADCVIVGGGISGL CIAQALVTKH Arabpt-1 RLRCSVAGGP TVGSSKIEGG GGT.TITTDCVIVGGGISGL CIAQALATKH Sorghumpt-1 .......... .......... .................... .......... Mzpt-1 .......... .......... .......ADC VVVGGGISGLCTAQALATRH Qtpt-1 RVRPRCATAS SATETPAAPG VRL...SAEC VIVGAGISGL CTAQALATRYRicept-1 .......... .......... .......... .......... ..........Cottonpt-1 KLRCSLAEGP TISSSKIDGG ESS...IADC VIVGGGISGL CIAQALATKHSoybeanpt1 ILRCSIAEES TASPPKTR.. DSA...PVDC VVVGGGVSGL CIAQALATKHSugpt-1 MSMSCSTSSG SKSAVKEAGS GSGAGGLLDC VIVGGGISGL CIAQALCTKH Scpt-1.......... .......... .......... .......... .......... 101 150 Rapept-1PDA..AKNVM VTEAKDRVGG NIIT..REEQ GFLWEEGPNS FQPSDPMLTM Arabpt-1PDA..APNLI VTEAKDRVGG NIIT..REEN GFLWEEGPNS FQPSDPMLTM Sorghumpt-1.......... .......... ..STVERPEE GYLWEEGPNS FQPSDPVLSM Mzpt-1 ..G..VGDVLVTEARARPGG NITTVERPEE GYLWEEGPNS FQPSDPVLTM Wtpt-1 ..G..VSDLL VTEARDRPGGNITTVERPDE GYLWEEGPNS FQPSDPVLTM Ricept-1 .......... .................... .......... .......... Cottonpt-1 RDV..ASNVI VTEARDRVGGNITTER..D GYLWEEGPNS FQPSDPILTM Soybeanpt1 ..A..NANVV VTEARDRVGGNITTMER..D GYLWEEGPNS FQPSDPMLTM Sugpt-1 SSSSLSPNFI VTEAKDRVGGNIVTVE..AD GYIWEEGPNS FQPSDAVLTM Scpt-1 .......... .......... .................... .......... 151 200 Rapept-1 VVDSGLKDDL VLGDPTAPRF VLWNGKLRPVPSKLTDLPFF DLMSIGGKIR Arabpt-1 VVDSGLKDDL VLGDPTAPRF VLWNGKLRPVPSKLTDLPFF DLMSIPGKIR Sorghumpt-1 AVDSGLKDDL VFGDPNAPRF VLWEGKLRPVPSKPADLPFF DLMSIPGKLR Mzpt-1 AVDSGLKDDL VFGDPNAPRF VLWEGKLRPV PSKPADLPFFDLMSIPGKLR Wtpt-1 AVDSGLKDDL VFGDPNAPRF VLWEGKLRPV PSKPADLPFF DLMSIPGKLRRicept-1 .......... .......... .......... .......... ..........Cottonpt-1 AVDSGLKDDL VLGDPNAPRF VLWEGKLRPV PSKPTDLPFF DLMSIAGKLRSoybeanpt1 VVDSGLKDEL VLGDPDARRF VLWNRKLRPV PGKLTDLPFF DLMSIGGKIRSugpt-1 AVDSGLKDEL VLGDPNAPRF VLWNDKLRPV PSSLTDLPFF DLMTIPGKIR Scpt-1.......... .......... .......... .......... .......... 201 250 Rapept-1AGFGAIGIRP SPPGREESVE EFVRRNLGDE VFERLIEPFC SGVYAGDPAK Arabpt-1AGFGALGIRP SPPGREESVE EFVRRNLGDE VFERLIEPFC SGVYAGDPSK Sorghumpt-1AGLGALGIRP PAPGREESVE EFVRRNLGAE VFERLIEPFC SGVYAGDPSK Mzpt-1 AGLGALGIRPPPPGREESVE EFVRRNLGAE VFERLIEPFC SGVYAGDPSK Wtpt-1 AGLGALGIRP PPPGREESVEEFVRRNLGAE VFERLIEPFC SGVYAGDPSK Ricept-1 .......... .................... .......... .......... Cottonpt-1 AGFGAIGIRP PPPGYEESVEEFVRRNLGAE VFERFIEPFC SGVYAGDPSK Soybeanpt1 AGFGALGIRP PPPGHEESVEEFVRRNLGDE VFERLIEPFC SGVYAGDPSK Sugpt-1 AALGALGFRP SPPPHEESVEHFVRRNLGDE VFERLIEPFC SGVYAGDPAK Scpt-1 .......... .......... .................... .......... 251 300 Rapept-1 LSMKAAFGKV WKLEENGGSI IGGAFKAIQAKNKAPKTTRD PRLPKPKGQT Arabpt-1 LSMKAAFGKV WKLEQNGGSI IGGTFKAIQERKNAPKAERD PRLPKPQGQT Sorghumpt-1 LSMKAAFGKV WRLEEAGGSI IGGTIKTIQERGKNPKPPRD PRLPKPKGQT Mzpt-1 LSMKAAFGKV WRLEETGGSI IGGTIKTIQE RSKNPKPPRDARLPKPKGQT Wtpt-1 LSMKAAFGKV WRLEEIGGSI IGGTIKAIQD KGKNPKPPRD PRLPAPKGQTRicept-1 RALKAAFGKV WRLEDTGGSI IGGTIKTIQE RGKNPKPPRD PRLPTPKGQTCottonpt-1 LSMKAAFGRV WKLLEIGGSI IGGTIKTIQE RNKTPKPPRD PRLPKPKGQTSoybeanpt1 LSMKAAFGKV WKLEKNGGSI IGGTFKAIQE RNGASKPPRD PRLPKPKGQTSugpt-1 LSMKAAFGKV WKLEQKGGSI IGGTLKAIQE RGSNPKPPRD QRLPKPKGQT Scpt-1.......... .......... .......... .......... .......... 301 350 Rapept-1VGSFRKGLTM LPEAISARLG DKVKVSWKLS SITKLASGEY SLTYETPEGI Arabpt-1VGSFRKGLRM LPEAISARLG SKVKLSWKLS GITKLESGGY NLTYETPDGL Sorghumpt-1VASFRKGLAM LPNAITSSLG SKVKLSWKLT SMTKSDGKGY VLEYETPEGV Mzpt-1 VASFRKGLAMLPNAITSSLG SKVKLSWKLT SITKSDDKGY VLEYETPEGV Wtpt-1 VASFRKGLAM LPNAIASRLGSKVKLSWKLT SITKADNQGY VLGYETPEGL Ricept-1 VASFRKGLTM LPDAITSRLGSKVKLSWKLT SITKSDNKGY ALVYETPEGV Cottonpt-1 VGSFRKGLTM LPEAIANSLGSNVKLSWKLS SIRKLGNGGY NLTFETPEGM Soybeanpt1 VGSFRKGLTM LPDAISARLGNKVKLSWKLS SITKLGNGEY SLTYETPEGV Sugpt-1 VGSFRKGLVM LPTAISARLGSRVKLSWTLS SIVKSLNGEY SLTYDTPDGL Scpt-1 .......... .......... .................... .......... 351 400 Rapept-1 VTVQSKSVVM TVPSHVASSL LRPLSDSAAEALSKLYYPPV AAVSISYAKE Arabpt-1 VSVQSKSVVM TVPSHVASGL LRPLSESAANALSKLYYPPV AAVSISYPKE Sorghumpt-1 VLVQAKSVIM TIPSSYVASDI LRPLSGDAADVLSRFYYPPV AAVTVSYPKE Mzpt-1 VSVQAKSVIM TIPSSTVVASNI LRPLSSDAADALDRFYYPPV AAVTVSYPKE Wtpt-1 VSVQAKSVIM TIPSYVASDI LRPLSIDAAD ALSKFYYPPVAAVTVSYPKE Ricept-1 VSVQAKTVVM TIPSYVASDI LRPLSSDAAD ALSIFYYPPVAAVTVSYPKE Cottonpt-1 VSLQSRSVVM TIPSHVASNL LHPLSAAAAD ALSQFYYPPVASVTVSYPKE Soybeanpt1 VSLQCKTVVL TIPSYVASTL LRPLSAAAAD ALSKFYYPPVAAVSISYPKE Sugpt-1 VSVRTKSVVM TVPSYVASRL LRPLSDSAAD SLSKFYYPPVAAVSLSYPKE Scpt-1 .......... .......... .......... .......... ..........401 450 Rapept-1 AIRSECLIDG ELKGFGQLHP RTQKVETLGT IYSSSLFPNR APPGRVLLLNArabpt-1 AIRTECLIDG ALKGFGQLHP RTQGVETLGT IYSSSLFPNR APPGRILLLNSorghumpt-1 AIRKECLIDG ELQGFGQLHP RSQGVETLGT IYSSSLFPNR APAGRVLLLNMzpt-1 AIRKECLIDG ELQGFGQLHP RSQGVETLGT IYSSSLFPNR APDGRVLLLN Wtpt-1AIRKECLIDG ELQGFGQLHP RSQGVETLGT IYSSSLFPNR APAGRVLLLN Ricept-1AIRKECLIDG ELQGFGQLHP RSQGVETLGT IYSSSLFPNR APAGRVLLLN Cottonpt-1AIRKECLIDG ELKGFGQLHP RSQGIETLGT IYSSSLFPNR APSGRVLLLN Soybeanpt1AIRSECLIDG ELKGFGQLHP RSQGVETLGT IYSSSLFPNR APPGRVLLLN Sugpt-1AIRSECLING ELQGFGQLHP RSQGVETLGT IYSSSLFPGR APPGRILILS 451 500 Rapept-1YIGGATNTGI LSKSEGELVE AVDRDLRKML IKPSSTDPLV LGVKLWPQAI Arabpt-1YIGGSTNTGI LSKSEGELVE AVDRDLRKML IKPNSTDPLK LGVRVWPQAI Sorghumpt-1YIGGATNTGI VSKTESELVE AVDRDLRKML INPTAVDPLV LGVRVWPQAI Mzpt-1 YIGGATNTGIVSKTESELVE AVDRDLRKML INSTAVDPLV LGVRVWPQAI Wtpt-1 YIGGSTNTGI VSKTESDLVGAVDRDLRKML INPRAADPLA LGVRVWPQAI Ricept-1 YIGGSTNTGI VSKTESELVEAVDRDLRKML INPNAKDPLV LGVRVWPKAI Soybeanpt1 YIGGATNTGI LSKTDSELVETVDRDLRKIL INPNAQDPFV VGVRLWPQAI Sugpt-1 YIGGAKNPGI LNKSKDElAKTVDKDLRRML INPDAKLPRV LGVRVWPQAI Scpt-1 .......... .SKTESELVE AVDRDLRKMLINPTAVDPLV LGVRVWPQAI 501 550 Rapept-1 PQFLIGHIDL VDAAKASLSS SGHEGLFLGGNYVAGVALGR CVEGAYETAT Arabpt-1 PQFLVGHFDI LDTAKSSLTS SGYEGLFLGGNYVAGVALGR CVEGAYETAI Sorghumpt-1 PQFLVGHLDL LEAAKSALDQ GGYBGLFLGGNYVAGVALGR CIEGAYESAA Mzpt-1 PQFLVGHLDL LEAAKAALDR GGYDGLFLGG NYVAGVALGRCVEGAYESAS Wtpt-1 PQFLIGHLDR LAAAKSALGQ GGYDGLFLGG KYVAGVALGR CIEGAYESASRicept-1 PQFLIGHLDH LEAAKSALGK GGYDGLFLGG NYVAGVALGR CVEGAYESASCottonpt-1 PQFLVGHLDL LDSAKMALRD SGFHGLFLGG NYVSGVALGR CVEGAYEVAASoybeanpt1 PQFLVGHLDL LDVAKASIRN TGFEGLFLGG NYVSGVALGR CVEGAYEVAASugpt-1 PQFSIGHFDL LDAAKAALTD TGVKGLFLGG NYVSGVALGR CIEGAYESAA Scpt-1PQFLVGHLDL LEAAKSALDR GGYDGLFLGG NYVAGVALGR CVEGAYESAS 551 563 Rapept-1QVNDFMSRYA YK* Arabpt-1 EVNNFMSRYA YK* Sorghumpt-1 QIYDFLTKYA YK* Mzpt-1QISDFLTKYA YK* Wtpt-1 QVSDFLTKYA YK* Ricept-1 QISDYLTKYA YK* Cottonpt-1EVKEFLSQYA YK* Soybeanpt1 EVNDFLTNRV YK* Sugpt-1 EVVDFLSQYS DK* Scpt-1QIYDFLTKYA YK*

[0591] TABLE 1B Sub-sequences of herbicide-tolerant protox enzymescomprising point mutations. Corresponding Δ_(n) AA AA position ofExemplary # Sub-sequence wild-type Δ_(n) AA substitutions Δ_(n) in Table1A nutants  1 APΔ₁F R C 169 Mz88Cys  2 FΔ₂S C F, L, K 240 Mz159PheMz159Leu Mz159Lys  3 YΔ₃G A V, T, L, C, I 245 pAraC-1Val pAraC-1ThrpAraC-1Leu pAraC-1Cys pAraC-1Ile pMzC-1Val pMzC-1Thr pMzC-1LeupWhtC-1Val pWhtC-1Thr pSoyC-1Thr pSoyC-1Leu  4 AΔ₄D S, L 246 pAraC-3SerpMzC-3Ser pMzC-3Leu pWhtC-3Ser  5 YΔ₅P P S, H 388 Soy369Ser Soy369HisCot365Ser  6 PΔ₆A V L 390 Wht356Leu  7 Δ₇IG Y C, I, L, T, M, V, 451pAraC-2Cys A, R pAraC-2Ile pAraC-2Leu pAraC-2Thr pAraC-2Met pAraC-2ValpAraC-2Ala pMzC-2Ile pMzC-2Met pSoyC-2Leu pSoyC-2Ile pSoyC-2IlepSugC-2Cys pSugC-2Leu pSugC-2Ile pSugC-2Val pSugC-2Met pCotC-2CyspCotC-2Arg  8 YIGGΔ₈ A, S P 455 Wht421Pro  9 AΔ₉P I T, H, G, N 500Mz419Thr Mz419His Mz419Gly Mz419Asn Wht466Thr 10 GΔ₁₀A V A 536 Wht502AlaSoy517Ala Second-site mutations 11 QΔ₁₁S P L 143 AraC118Leu 12 IGGΔ₁₂ TI, A 274 AraC249Ile AraC249Ala 13 SWXLΔ₁₃ S, T L 330 AraC305Leu 14 LΔ₁₄YN S 450 AraC425Ser 15 GΔ₁₅XGL Y, H, F, V C 523 AraC498Cys Doublemutation 16 TΔ₁₆G L S 428 Mz347Ser453Thr 17 YVΔ₁₇G A, (S) T 534

[0592] TABLE 2 Comparison of the Arabidopsis (SEQ ID NO:4) and maize(SEQ ID NO:8) protox-2 amino acid sequences. Identical residues aredenoted by the vertical bars between the two sequences. Alignment wasperformed using the GAP program described in Deveraux et al., NucleicAcids Res. 12:387-395 (1984). Percent similarity: 75.889/percentidentity: 57.905. 1 ...........................MASGAVAD.HQIEAVSGKRVAV 21                            .|  |:|: .:  |..::.||| 1MLALTASASSASSHPYRHASAHTRRPRLRAVLAMAGSDDPRAAPARSVAV 50 22VGAGVSGLAAAYKLKSRGLNVTVFEADGRVGGKLRSVMQNGLIWDEGANT 71||||||||||||:|: .|:|||||||.:|.|||:|.  :.|::||||||| 51VGAGVSGLAAAYRLRQSGNVTVFEAADRAGGKIRTNSEGGFVWDEGANT 100 72MTEAEPEVGSLLDDLGLREKQQFPISQKKRYIVRNGVPVMLPTNPIELVT 121|||:| |.:.|:|||||.:|||:★ ||.|||||::|.|.::|.:||.|:. 101MTEGEWEASRLIDDLGLQDKQQYPNSQHKRIYVKDGAPALIPSDPISLMK 150 122SSVLSTQSKFQILLEPFLWKK....KSSKVSDASAEESVSEFFQRHFGQE 167||||||.||:.:::||||:||    .|:|||:.  .|||:.| :||||.| 151SSVLSTKSKIALFFEPFLYKKANTRNSGKVSEEHLSESVGSFCERHFGRE 200 168VVDYLIDPFVGGTSAADPDSLSMKHSFPDLWNVEKSFGSIIVGAIRTKFA 217||||::||||:||||:||:|||::|.||.|||:|:.:||:||||| .|:| 201VVDYFVDPFVAGTSAGDPESLSIRHAFPALWNLERKYGSVIVGAILSKLA 250 218AKGGKSRDTKSSPGTKKGSRGSFSFKGGMQILPDTLCKSLSHDEINKDSK 267|||:. :. ..|.|.::..|.||||.|||| | :.| ..::.|::.|:.. 251AKGSPVKTRHDSSGKRRNRRVSFSFHGGMQSLINALHNEVGDDNVKLGTE 300 268VLSLS..YNSGSRQENWSLSCVSHNETQRQ...NPHYDAVIMTAPLCNVK 312||||.  :::..  :.||:|. |.:..:::   |. :|||||||| :||: 301VLSLACTFDGVPALGRWSISVDSKDSGDKDLASNQTFDAVIMTAPLSNVR 350 313EMKVMKGGQPFQLNFLPEINYMPLSVLITTFTKEKVKRPLEGFGVLIPSK 362 ||. |||.|. |:|||.::|:|||:::|.|.|:.||:|||||||||| | 351RMKFTKGGAPVVLDFLPKMSYLPLSLMVTAFKKDDVKKPLEGFGVLIPYK 400 363E.QKHGFKTLGTLFSSMMFPDRSPSDVHLYTTFIGGSRNQELAKASTDEL 411| ||||:|||||||||||||||.|.|.| .|||||:|||:|.:|| |.|. | 401EQQKHGLKTLGTLFSSMMFPDRAPDDQYLYTTFVGGSHNRDLAGAPTSIL 450 412KQVVTSDLQRLLGVEGEPVSVNHYYWRKAFPLYDSSYDSVMEAIDKMEND 461||:|||||.:||||||:|. |. |.| || .|||||: .|.||:|||:|||.: 451KQLVTSDLKKLLGVEGQPTFVKHVYWGNAFPLYGHDYSSVLEAIEKMEKN 500 462LPGFFYAGNHRGGLSVGKSIASGCKAADLVISYLESCSNDKKPNDSL* 509||||||||| ::||.||. ||||:|||||.|||||| ......: 501LPGFFYAGNSKDGLAVGSVIASGSKAADLAISYLESHTKHNNSH*... 545

[0593] TABLE 3A Cross tolerance of plant protox mutants to variousprotox inhibitors. Formula AraC-1 Val AraC-2Cys AraC-1Thr AraC-3ThrMzC-1Val XVII + + + + + VIIa + + + − + IV ++ − ++ ++ − XV + + + + + XI− + + ++ + XVI − − − − + XII + − ++ ++ ++ XIV + − + + + *X

[0594] TABLE 3B Cross tolerance of plant protox mutants to variousprotox inhibitors. AraC- AraC- AraC- AraC- AraC- AraC- 1Leu 1Leu 2Ile2Cys 2Leu 2Met + + + + + + AraC- AraC- AraC- AraC- AraC AraC AraC AraCFormula 1Leu 2Ile 2Met 2Leu 305Leu 425Ser 425Ser 425SerXVII + + + + + + + + VIIa ++ ++ ++ ++ ++ ++ ++ ++ IV ++ − + ++ + − + +XV ++ +++ +++ +++ +++ ++ +++ ++ XI ++ ++ ++ ++ ++ ++ ++ ++ XVI +++ ++++++ +++ +++ + ++ ++ XII XIV ++ ++ ++ ++ ++ − ++ ++

[0595] TABLE 4 Cross tolerance to various protox inhibitors in a seedgermination assay. Formula Common name Tolerance II acifluorofen + IIIfomasafen + IV fluoroglycofen ± IVb bifenox + IVc oxyfluorofen + IVdlactofen ± VIIa fluthiacet-methyl ++ X sulfentrazone + XI flupropazil ++XIV flumiclorac + XVI flumioxazin +++ XVII ++ XXIa BAY 11340 + XXII ++

[0596] Various modifications of the invention described herein willbecome apparent to those skilled in the art. Such modifications areintended to fall within the scope of the appended claims.

1 37 1719 base pairs nucleic acid single linear cDNA NO NO Arabidopsisthaliana pWDC-2 (NRRL B-21238) CDS 31..1644 /product= “Arabidopsisprotox-1” 1 TGACAAAATT CCGAATTCTC TGCGATTTCC ATG GAG TTA TCT CTT CTC CGTCCG 54 Met Glu Leu Ser Leu Leu Arg Pro 1 5 ACG ACT CAA TCG CTT CTT CCGTCG TTT TCG AAG CCC AAT CTC CGA TTA 102 Thr Thr Gln Ser Leu Leu Pro SerPhe Ser Lys Pro Asn Leu Arg Leu 10 15 20 AAT GTT TAT AAG CCT CTT AGA CTCCGT TGT TCA GTG GCC GGT GGA CCA 150 Asn Val Tyr Lys Pro Leu Arg Leu ArgCys Ser Val Ala Gly Gly Pro 25 30 35 40 ACC GTC GGA TCT TCA AAA ATC GAAGGC GGA GGA GGC ACC ACC ATC ACG 198 Thr Val Gly Ser Ser Lys Ile Glu GlyGly Gly Gly Thr Thr Ile Thr 45 50 55 ACG GAT TGT GTG ATT GTC GGC GGA GGTATT AGT GGT CTT TGC ATC GCT 246 Thr Asp Cys Val Ile Val Gly Gly Gly IleSer Gly Leu Cys Ile Ala 60 65 70 CAG GCG CTT GCT ACT AAG CAT CCT GAT GCTGCT CCG AAT TTA ATT GTG 294 Gln Ala Leu Ala Thr Lys His Pro Asp Ala AlaPro Asn Leu Ile Val 75 80 85 ACC GAG GCT AAG GAT CGT GTT GGA GGC AAC ATTATC ACT CGT GAA GAG 342 Thr Glu Ala Lys Asp Arg Val Gly Gly Asn Ile IleThr Arg Glu Glu 90 95 100 AAT GGT TTT CTC TGG GAA GAA GGT CCC AAT AGTTTT CAA CCG TCT GAT 390 Asn Gly Phe Leu Trp Glu Glu Gly Pro Asn Ser PheGln Pro Ser Asp 105 110 115 120 CCT ATG CTC ACT ATG GTG GTA GAT AGT GGTTTG AAG GAT GAT TTG GTG 438 Pro Met Leu Thr Met Val Val Asp Ser Gly LeuLys Asp Asp Leu Val 125 130 135 TTG GGA GAT CCT ACT GCG CCA AGG TTT GTGTTG TGG AAT GGG AAA TTG 486 Leu Gly Asp Pro Thr Ala Pro Arg Phe Val LeuTrp Asn Gly Lys Leu 140 145 150 AGG CCG GTT CCA TCG AAG CTA ACA GAC TTACCG TTC TTT GAT TTG ATG 534 Arg Pro Val Pro Ser Lys Leu Thr Asp Leu ProPhe Phe Asp Leu Met 155 160 165 AGT ATT GGT GGG AAG ATT AGA GCT GGT TTTGGT GCA CTT GGC ATT CGA 582 Ser Ile Gly Gly Lys Ile Arg Ala Gly Phe GlyAla Leu Gly Ile Arg 170 175 180 CCG TCA CCT CCA GGT CGT GAA GAA TCT GTGGAG GAG TTT GTA CGG CGT 630 Pro Ser Pro Pro Gly Arg Glu Glu Ser Val GluGlu Phe Val Arg Arg 185 190 195 200 AAC CTC GGT GAT GAG GTT TTT GAG CGCCTG ATT GAA CCG TTT TGT TCA 678 Asn Leu Gly Asp Glu Val Phe Glu Arg LeuIle Glu Pro Phe Cys Ser 205 210 215 GGT GTT TAT GCT GGT GAT CCT TCA AAACTG AGC ATG AAA GCA GCG TTT 726 Gly Val Tyr Ala Gly Asp Pro Ser Lys LeuSer Met Lys Ala Ala Phe 220 225 230 GGG AAG GTT TGG AAA CTA GAG CAA AATGGT GGA AGC ATA ATA GGT GGT 774 Gly Lys Val Trp Lys Leu Glu Gln Asn GlyGly Ser Ile Ile Gly Gly 235 240 245 ACT TTT AAG GCA ATT CAG GAG AGG AAAAAC GCT CCC AAG GCA GAA CGA 822 Thr Phe Lys Ala Ile Gln Glu Arg Lys AsnAla Pro Lys Ala Glu Arg 250 255 260 GAC CCG CGC CTG CCA AAA CCA CAG GGCCAA ACA GTT GGT TCT TTC AGG 870 Asp Pro Arg Leu Pro Lys Pro Gln Gly GlnThr Val Gly Ser Phe Arg 265 270 275 280 AAG GGA CTT CGA ATG TTG CCA GAAGCA ATA TCT GCA AGA TTA GGT AGC 918 Lys Gly Leu Arg Met Leu Pro Glu AlaIle Ser Ala Arg Leu Gly Ser 285 290 295 AAA GTT AAG TTG TCT TGG AAG CTCTCA GGT ATC ACT AAG CTG GAG AGC 966 Lys Val Lys Leu Ser Trp Lys Leu SerGly Ile Thr Lys Leu Glu Ser 300 305 310 GGA GGA TAC AAC TTA ACA TAT GAGACT CCA GAT GGT TTA GTT TCC GTG 1014 Gly Gly Tyr Asn Leu Thr Tyr Glu ThrPro Asp Gly Leu Val Ser Val 315 320 325 CAG AGC AAA AGT GTT GTA ATG ACGGTG CCA TCT CAT GTT GCA AGT GGT 1062 Gln Ser Lys Ser Val Val Met Thr ValPro Ser His Val Ala Ser Gly 330 335 340 CTC TTG CGC CCT CTT TCT GAA TCTGCT GCA AAT GCA CTC TCA AAA CTA 1110 Leu Leu Arg Pro Leu Ser Glu Ser AlaAla Asn Ala Leu Ser Lys Leu 345 350 355 360 TAT TAC CCA CCA GTT GCA GCAGTA TCT ATC TCG TAC CCG AAA GAA GCA 1158 Tyr Tyr Pro Pro Val Ala Ala ValSer Ile Ser Tyr Pro Lys Glu Ala 365 370 375 ATC CGA ACA GAA TGT TTG ATAGAT GGT GAA CTA AAG GGT TTT GGG CAA 1206 Ile Arg Thr Glu Cys Leu Ile AspGly Glu Leu Lys Gly Phe Gly Gln 380 385 390 TTG CAT CCA CGC ACG CAA GGAGTT GAA ACA TTA GGA ACT ATC TAC AGC 1254 Leu His Pro Arg Thr Gln Gly ValGlu Thr Leu Gly Thr Ile Tyr Ser 395 400 405 TCC TCA CTC TTT CCA AAT CGCGCA CCG CCC GGA AGA ATT TTG CTG TTG 1302 Ser Ser Leu Phe Pro Asn Arg AlaPro Pro Gly Arg Ile Leu Leu Leu 410 415 420 AAC TAC ATT GGC GGG TCT ACAAAC ACC GGA ATT CTG TCC AAG TCT GAA 1350 Asn Tyr Ile Gly Gly Ser Thr AsnThr Gly Ile Leu Ser Lys Ser Glu 425 430 435 440 GGT GAG TTA GTG GAA GCAGTT GAC AGA GAT TTG AGG AAA ATG CTA ATT 1398 Gly Glu Leu Val Glu Ala ValAsp Arg Asp Leu Arg Lys Met Leu Ile 445 450 455 AAG CCT AAT TCG ACC GATCCA CTT AAA TTA GGA GTT AGG GTA TGG CCT 1446 Lys Pro Asn Ser Thr Asp ProLeu Lys Leu Gly Val Arg Val Trp Pro 460 465 470 CAA GCC ATT CCT CAG TTTCTA GTT GGT CAC TTT GAT ATC CTT GAC ACG 1494 Gln Ala Ile Pro Gln Phe LeuVal Gly His Phe Asp Ile Leu Asp Thr 475 480 485 GCT AAA TCA TCT CTA ACGTCT TCG GGC TAC GAA GGG CTA TTT TTG GGT 1542 Ala Lys Ser Ser Leu Thr SerSer Gly Tyr Glu Gly Leu Phe Leu Gly 490 495 500 GGC AAT TAC GTC GCT GGTGTA GCC TTA GGC CGG TGT GTA GAA GGC GCA 1590 Gly Asn Tyr Val Ala Gly ValAla Leu Gly Arg Cys Val Glu Gly Ala 505 510 515 520 TAT GAA ACC GCG ATTGAG GTC AAC AAC TTC ATG TCA CGG TAC GCT TAC 1638 Tyr Glu Thr Ala Ile GluVal Asn Asn Phe Met Ser Arg Tyr Ala Tyr 525 530 535 AAG TAAATGTAAAACATTAAATC TCCCAGCTTG CGTGAGTTTT ATTAAATATT 1691 Lys TTGAGATATCCAAAAAAAAA AAAAAAAA 1719 537 amino acids amino acid linear protein notprovided 2 Met Glu Leu Ser Leu Leu Arg Pro Thr Thr Gln Ser Leu Leu ProSer 1 5 10 15 Phe Ser Lys Pro Asn Leu Arg Leu Asn Val Tyr Lys Pro LeuArg Leu 20 25 30 Arg Cys Ser Val Ala Gly Gly Pro Thr Val Gly Ser Ser LysIle Glu 35 40 45 Gly Gly Gly Gly Thr Thr Ile Thr Thr Asp Cys Val Ile ValGly Gly 50 55 60 Gly Ile Ser Gly Leu Cys Ile Ala Gln Ala Leu Ala Thr LysHis Pro 65 70 75 80 Asp Ala Ala Pro Asn Leu Ile Val Thr Glu Ala Lys AspArg Val Gly 85 90 95 Gly Asn Ile Ile Thr Arg Glu Glu Asn Gly Phe Leu TrpGlu Glu Gly 100 105 110 Pro Asn Ser Phe Gln Pro Ser Asp Pro Met Leu ThrMet Val Val Asp 115 120 125 Ser Gly Leu Lys Asp Asp Leu Val Leu Gly AspPro Thr Ala Pro Arg 130 135 140 Phe Val Leu Trp Asn Gly Lys Leu Arg ProVal Pro Ser Lys Leu Thr 145 150 155 160 Asp Leu Pro Phe Phe Asp Leu MetSer Ile Gly Gly Lys Ile Arg Ala 165 170 175 Gly Phe Gly Ala Leu Gly IleArg Pro Ser Pro Pro Gly Arg Glu Glu 180 185 190 Ser Val Glu Glu Phe ValArg Arg Asn Leu Gly Asp Glu Val Phe Glu 195 200 205 Arg Leu Ile Glu ProPhe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser 210 215 220 Lys Leu Ser MetLys Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Gln 225 230 235 240 Asn GlyGly Ser Ile Ile Gly Gly Thr Phe Lys Ala Ile Gln Glu Arg 245 250 255 LysAsn Ala Pro Lys Ala Glu Arg Asp Pro Arg Leu Pro Lys Pro Gln 260 265 270Gly Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Arg Met Leu Pro Glu 275 280285 Ala Ile Ser Ala Arg Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu 290295 300 Ser Gly Ile Thr Lys Leu Glu Ser Gly Gly Tyr Asn Leu Thr Tyr Glu305 310 315 320 Thr Pro Asp Gly Leu Val Ser Val Gln Ser Lys Ser Val ValMet Thr 325 330 335 Val Pro Ser His Val Ala Ser Gly Leu Leu Arg Pro LeuSer Glu Ser 340 345 350 Ala Ala Asn Ala Leu Ser Lys Leu Tyr Tyr Pro ProVal Ala Ala Val 355 360 365 Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg ThrGlu Cys Leu Ile Asp 370 375 380 Gly Glu Leu Lys Gly Phe Gly Gln Leu HisPro Arg Thr Gln Gly Val 385 390 395 400 Glu Thr Leu Gly Thr Ile Tyr SerSer Ser Leu Phe Pro Asn Arg Ala 405 410 415 Pro Pro Gly Arg Ile Leu LeuLeu Asn Tyr Ile Gly Gly Ser Thr Asn 420 425 430 Thr Gly Ile Leu Ser LysSer Glu Gly Glu Leu Val Glu Ala Val Asp 435 440 445 Arg Asp Leu Arg LysMet Leu Ile Lys Pro Asn Ser Thr Asp Pro Leu 450 455 460 Lys Leu Gly ValArg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Val 465 470 475 480 Gly HisPhe Asp Ile Leu Asp Thr Ala Lys Ser Ser Leu Thr Ser Ser 485 490 495 GlyTyr Glu Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala 500 505 510Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala Ile Glu Val Asn 515 520525 Asn Phe Met Ser Arg Tyr Ala Tyr Lys 530 535 1738 base pairs nucleicacid single linear cDNA NO NO Arabidopsis thaliana pWDC-1 (NRRL B-21237)CDS 70..1596 /product= “Arabidopsis protox-2” 3 TTTTTTACTT ATTTCCGTCACTGCTTTCGA CTGGTCAGAG ATTTTGACTC TGAATTGTTG 60 CAGATAGCA ATG GCG TCT GGAGCA GTA GCA GAT CAT CAA ATT GAA GCG 108 Met Ala Ser Gly Ala Val Ala AspHis Gln Ile Glu Ala 1 5 10 GTT TCA GGA AAA AGA GTC GCA GTC GTA GGT GCAGGT GTA AGT GGA CTT 156 Val Ser Gly Lys Arg Val Ala Val Val Gly Ala GlyVal Ser Gly Leu 15 20 25 GCG GCG GCT TAC AAG TTG AAA TCG AGG GGT TTG AATGTG ACT GTG TTT 204 Ala Ala Ala Tyr Lys Leu Lys Ser Arg Gly Leu Asn ValThr Val Phe 30 35 40 45 GAA GCT GAT GGA AGA GTA GGT GGG AAG TTG AGA AGTGTT ATG CAA AAT 252 Glu Ala Asp Gly Arg Val Gly Gly Lys Leu Arg Ser ValMet Gln Asn 50 55 60 GGT TTG ATT TGG GAT GAA GGA GCA AAC ACC ATG ACT GAGGCT GAG CCA 300 Gly Leu Ile Trp Asp Glu Gly Ala Asn Thr Met Thr Glu AlaGlu Pro 65 70 75 GAA GTT GGG AGT TTA CTT GAT GAT CTT GGG CTT CGT GAG AAACAA CAA 348 Glu Val Gly Ser Leu Leu Asp Asp Leu Gly Leu Arg Glu Lys GlnGln 80 85 90 TTT CCA ATT TCA CAG AAA AAG CGG TAT ATT GTG CGG AAT GGT GTACCT 396 Phe Pro Ile Ser Gln Lys Lys Arg Tyr Ile Val Arg Asn Gly Val Pro95 100 105 GTG ATG CTA CCT ACC AAT CCC ATA GAG CTG GTC ACA AGT AGT GTGCTC 444 Val Met Leu Pro Thr Asn Pro Ile Glu Leu Val Thr Ser Ser Val Leu110 115 120 125 TCT ACC CAA TCT AAG TTT CAA ATC TTG TTG GAA CCA TTT TTATGG AAG 492 Ser Thr Gln Ser Lys Phe Gln Ile Leu Leu Glu Pro Phe Leu TrpLys 130 135 140 AAA AAG TCC TCA AAA GTC TCA GAT GCA TCT GCT GAA GAA AGTGTA AGC 540 Lys Lys Ser Ser Lys Val Ser Asp Ala Ser Ala Glu Glu Ser ValSer 145 150 155 GAG TTC TTT CAA CGC CAT TTT GGA CAA GAG GTT GTT GAC TATCTC ATC 588 Glu Phe Phe Gln Arg His Phe Gly Gln Glu Val Val Asp Tyr LeuIle 160 165 170 GAC CCT TTT GTT GGT GGA ACA AGT GCT GCG GAC CCT GAT TCCCTT TCA 636 Asp Pro Phe Val Gly Gly Thr Ser Ala Ala Asp Pro Asp Ser LeuSer 175 180 185 ATG AAG CAT TCT TTC CCA GAT CTC TGG AAT GTA GAG AAA AGTTTT GGC 684 Met Lys His Ser Phe Pro Asp Leu Trp Asn Val Glu Lys Ser PheGly 190 195 200 205 TCT ATT ATA GTC GGT GCA ATC AGA ACA AAG TTT GCT GCTAAA GGT GGT 732 Ser Ile Ile Val Gly Ala Ile Arg Thr Lys Phe Ala Ala LysGly Gly 210 215 220 AAA AGT AGA GAC ACA AAG AGT TCT CCT GGC ACA AAA AAGGGT TCG CGT 780 Lys Ser Arg Asp Thr Lys Ser Ser Pro Gly Thr Lys Lys GlySer Arg 225 230 235 GGG TCA TTC TCT TTT AAG GGG GGA ATG CAG ATT CTT CCTGAT ACG TTG 828 Gly Ser Phe Ser Phe Lys Gly Gly Met Gln Ile Leu Pro AspThr Leu 240 245 250 TGC AAA AGT CTC TCA CAT GAT GAG ATC AAT TTA GAC TCCAAG GTA CTC 876 Cys Lys Ser Leu Ser His Asp Glu Ile Asn Leu Asp Ser LysVal Leu 255 260 265 TCT TTG TCT TAC AAT TCT GGA TCA AGA CAG GAG AAC TGGTCA TTA TCT 924 Ser Leu Ser Tyr Asn Ser Gly Ser Arg Gln Glu Asn Trp SerLeu Ser 270 275 280 285 TGT GTT TCG CAT AAT GAA ACG CAG AGA CAA AAC CCCCAT TAT GAT GCT 972 Cys Val Ser His Asn Glu Thr Gln Arg Gln Asn Pro HisTyr Asp Ala 290 295 300 GTA ATT ATG ACG GCT CCT CTG TGC AAT GTG AAG GAGATG AAG GTT ATG 1020 Val Ile Met Thr Ala Pro Leu Cys Asn Val Lys Glu MetLys Val Met 305 310 315 AAA GGA GGA CAA CCC TTT CAG CTA AAC TTT CTC CCCGAG ATT AAT TAC 1068 Lys Gly Gly Gln Pro Phe Gln Leu Asn Phe Leu Pro GluIle Asn Tyr 320 325 330 ATG CCC CTC TCG GTT TTA ATC ACC ACA TTC ACA AAGGAG AAA GTA AAG 1116 Met Pro Leu Ser Val Leu Ile Thr Thr Phe Thr Lys GluLys Val Lys 335 340 345 AGA CCT CTT GAA GGC TTT GGG GTA CTC ATT CCA TCTAAG GAG CAA AAG 1164 Arg Pro Leu Glu Gly Phe Gly Val Leu Ile Pro Ser LysGlu Gln Lys 350 355 360 365 CAT GGT TTC AAA ACT CTA GGT ACA CTT TTT TCATCA ATG ATG TTT CCA 1212 His Gly Phe Lys Thr Leu Gly Thr Leu Phe Ser SerMet Met Phe Pro 370 375 380 GAT CGT TCC CCT AGT GAC GTT CAT CTA TAT ACAACT TTT ATT GGT GGG 1260 Asp Arg Ser Pro Ser Asp Val His Leu Tyr Thr ThrPhe Ile Gly Gly 385 390 395 AGT AGG AAC CAG GAA CTA GCC AAA GCT TCC ACTGAC GAA TTA AAA CAA 1308 Ser Arg Asn Gln Glu Leu Ala Lys Ala Ser Thr AspGlu Leu Lys Gln 400 405 410 GTT GTG ACT TCT GAC CTT CAG CGA CTG TTG GGGGTT GAA GGT GAA CCC 1356 Val Val Thr Ser Asp Leu Gln Arg Leu Leu Gly ValGlu Gly Glu Pro 415 420 425 GTG TCT GTC AAC CAT TAC TAT TGG AGG AAA GCATTC CCG TTG TAT GAC 1404 Val Ser Val Asn His Tyr Tyr Trp Arg Lys Ala PhePro Leu Tyr Asp 430 435 440 445 AGC AGC TAT GAC TCA GTC ATG GAA GCA ATTGAC AAG ATG GAG AAT GAT 1452 Ser Ser Tyr Asp Ser Val Met Glu Ala Ile AspLys Met Glu Asn Asp 450 455 460 CTA CCT GGG TTC TTC TAT GCA GGT AAT CATCGA GGG GGG CTC TCT GTT 1500 Leu Pro Gly Phe Phe Tyr Ala Gly Asn His ArgGly Gly Leu Ser Val 465 470 475 GGG AAA TCA ATA GCA TCA GGT TGC AAA GCAGCT GAC CTT GTG ATC TCA 1548 Gly Lys Ser Ile Ala Ser Gly Cys Lys Ala AlaAsp Leu Val Ile Ser 480 485 490 TAC CTG GAG TCT TGC TCA AAT GAC AAG AAACCA AAT GAC AGC TTA TAA 1603 Tyr Leu Glu Ser Cys Ser Asn Asp Lys Lys ProAsn Asp Ser Leu 495 500 505 AAGGTTCGTC CCTTTTTATC ACTTACTTTG TAAACTTGTAAAATGCAACA AGCCGCCG 1663 CGATTAGCCA ACAACTCAGC AAAACCCAGA TTCTCATAAGGCTCACTAAT TCCAGAAT 1723 ACTATTTATG TAAAA 1738 508 amino acids aminoacid linear protein not provided 4 Met Ala Ser Gly Ala Val Ala Asp HisGln Ile Glu Ala Val Ser Gly 1 5 10 15 Lys Arg Val Ala Val Val Gly AlaGly Val Ser Gly Leu Ala Ala Ala 20 25 30 Tyr Lys Leu Lys Ser Arg Gly LeuAsn Val Thr Val Phe Glu Ala Asp 35 40 45 Gly Arg Val Gly Gly Lys Leu ArgSer Val Met Gln Asn Gly Leu Ile 50 55 60 Trp Asp Glu Gly Ala Asn Thr MetThr Glu Ala Glu Pro Glu Val Gly 65 70 75 80 Ser Leu Leu Asp Asp Leu GlyLeu Arg Glu Lys Gln Gln Phe Pro Ile 85 90 95 Ser Gln Lys Lys Arg Tyr IleVal Arg Asn Gly Val Pro Val Met Leu 100 105 110 Pro Thr Asn Pro Ile GluLeu Val Thr Ser Ser Val Leu Ser Thr Gln 115 120 125 Ser Lys Phe Gln IleLeu Leu Glu Pro Phe Leu Trp Lys Lys Lys Ser 130 135 140 Ser Lys Val SerAsp Ala Ser Ala Glu Glu Ser Val Ser Glu Phe Phe 145 150 155 160 Gln ArgHis Phe Gly Gln Glu Val Val Asp Tyr Leu Ile Asp Pro Phe 165 170 175 ValGly Gly Thr Ser Ala Ala Asp Pro Asp Ser Leu Ser Met Lys His 180 185 190Ser Phe Pro Asp Leu Trp Asn Val Glu Lys Ser Phe Gly Ser Ile Ile 195 200205 Val Gly Ala Ile Arg Thr Lys Phe Ala Ala Lys Gly Gly Lys Ser Arg 210215 220 Asp Thr Lys Ser Ser Pro Gly Thr Lys Lys Gly Ser Arg Gly Ser Phe225 230 235 240 Ser Phe Lys Gly Gly Met Gln Ile Leu Pro Asp Thr Leu CysLys Ser 245 250 255 Leu Ser His Asp Glu Ile Asn Leu Asp Ser Lys Val LeuSer Leu Ser 260 265 270 Tyr Asn Ser Gly Ser Arg Gln Glu Asn Trp Ser LeuSer Cys Val Ser 275 280 285 His Asn Glu Thr Gln Arg Gln Asn Pro His TyrAsp Ala Val Ile Met 290 295 300 Thr Ala Pro Leu Cys Asn Val Lys Glu MetLys Val Met Lys Gly Gly 305 310 315 320 Gln Pro Phe Gln Leu Asn Phe LeuPro Glu Ile Asn Tyr Met Pro Leu 325 330 335 Ser Val Leu Ile Thr Thr PheThr Lys Glu Lys Val Lys Arg Pro Leu 340 345 350 Glu Gly Phe Gly Val LeuIle Pro Ser Lys Glu Gln Lys His Gly Phe 355 360 365 Lys Thr Leu Gly ThrLeu Phe Ser Ser Met Met Phe Pro Asp Arg Ser 370 375 380 Pro Ser Asp ValHis Leu Tyr Thr Thr Phe Ile Gly Gly Ser Arg Asn 385 390 395 400 Gln GluLeu Ala Lys Ala Ser Thr Asp Glu Leu Lys Gln Val Val Thr 405 410 415 SerAsp Leu Gln Arg Leu Leu Gly Val Glu Gly Glu Pro Val Ser Val 420 425 430Asn His Tyr Tyr Trp Arg Lys Ala Phe Pro Leu Tyr Asp Ser Ser Tyr 435 440445 Asp Ser Val Met Glu Ala Ile Asp Lys Met Glu Asn Asp Leu Pro Gly 450455 460 Phe Phe Tyr Ala Gly Asn His Arg Gly Gly Leu Ser Val Gly Lys Ser465 470 475 480 Ile Ala Ser Gly Cys Lys Ala Ala Asp Leu Val Ile Ser TyrLeu Glu 485 490 495 Ser Cys Ser Asn Asp Lys Lys Pro Asn Asp Ser Leu 500505 1691 base pairs nucleic acid single linear cDNA NO NO Zea mays(maize) pWDC-4 (NRRL B-21260) CDS 1..1443 /product= “Maize protox-1 cDNA(not full-length); first seven nucleotides removed vs. serial no.60/012,705” 5 GCG GAC TGC GTC GTG GTG GGC GGA GGC ATC AGT GGC CTC TGCACC GCG 48 Ala Asp Cys Val Val Val Gly Gly Gly Ile Ser Gly Leu Cys ThrAla 1 5 10 15 CAG GCG CTG GCC ACG CGG CAC GGC GTC GGG GAC GTG CTT GTCACG GAG 96 Gln Ala Leu Ala Thr Arg His Gly Val Gly Asp Val Leu Val ThrGlu 20 25 30 GCC CGC GCC CGC CCC GGC GGC AAC ATT ACC ACC GTC GAG CGC CCCGAG 144 Ala Arg Ala Arg Pro Gly Gly Asn Ile Thr Thr Val Glu Arg Pro Glu35 40 45 GAA GGG TAC CTC TGG GAG GAG GGT CCC AAC AGC TTC CAG CCC TCC GAC192 Glu Gly Tyr Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp 5055 60 CCC GTT CTC ACC ATG GCC GTG GAC AGC GGA CTG AAG GAT GAC TTG GTT240 Pro Val Leu Thr Met Ala Val Asp Ser Gly Leu Lys Asp Asp Leu Val 6570 75 80 TTT GGG GAC CCA AAC GCG CCG CGT TTC GTG CTG TGG GAG GGG AAG CTG288 Phe Gly Asp Pro Asn Ala Pro Arg Phe Val Leu Trp Glu Gly Lys Leu 8590 95 AGG CCC GTG CCA TCC AAG CCC GCC GAC CTC CCG TTC TTC GAT CTC ATG336 Arg Pro Val Pro Ser Lys Pro Ala Asp Leu Pro Phe Phe Asp Leu Met 100105 110 AGC ATC CCA GGG AAG CTC AGG GCC GGT CTA GGC GCG CTT GGC ATC CGC384 Ser Ile Pro Gly Lys Leu Arg Ala Gly Leu Gly Ala Leu Gly Ile Arg 115120 125 CCG CCT CCT CCA GGC CGC GAA GAG TCA GTG GAG GAG TTC GTG CGC CGC432 Pro Pro Pro Pro Gly Arg Glu Glu Ser Val Glu Glu Phe Val Arg Arg 130135 140 AAC CTC GGT GCT GAG GTC TTT GAG CGC CTC ATT GAG CCT TTC TGC TCA480 Asn Leu Gly Ala Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser 145150 155 160 GGT GTC TAT GCT GGT GAT CCT TCT AAG CTC AGC ATG AAG GCT GCATTT 528 Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe165 170 175 GGG AAG GTT TGG CGG TTG GAA GAA ACT GGA GGT AGT ATT ATT GGTGGA 576 Gly Lys Val Trp Arg Leu Glu Glu Thr Gly Gly Ser Ile Ile Gly Gly180 185 190 ACC ATC AAG ACA ATT CAG GAG AGG AGC AAG AAT CCA AAA CCA CCGAGG 624 Thr Ile Lys Thr Ile Gln Glu Arg Ser Lys Asn Pro Lys Pro Pro Arg195 200 205 GAT GCC CGC CTT CCG AAG CCA AAA GGG CAG ACA GTT GCA TCT TTCAGG 672 Asp Ala Arg Leu Pro Lys Pro Lys Gly Gln Thr Val Ala Ser Phe Arg210 215 220 AAG GGT CTT GCC ATG CTT CCA AAT GCC ATT ACA TCC AGC TTG GGTAGT 720 Lys Gly Leu Ala Met Leu Pro Asn Ala Ile Thr Ser Ser Leu Gly Ser225 230 235 240 AAA GTC AAA CTA TCA TGG AAA CTC ACG AGC ATT ACA AAA TCAGAT GAC 768 Lys Val Lys Leu Ser Trp Lys Leu Thr Ser Ile Thr Lys Ser AspAsp 245 250 255 AAG GGA TAT GTT TTG GAG TAT GAA ACG CCA GAA GGG GTT GTTTCG GTG 816 Lys Gly Tyr Val Leu Glu Tyr Glu Thr Pro Glu Gly Val Val SerVal 260 265 270 CAG GCT AAA AGT GTT ATC ATG ACT ATT CCA TCA TAT GTT GCTAGC AAC 864 Gln Ala Lys Ser Val Ile Met Thr Ile Pro Ser Tyr Val Ala SerAsn 275 280 285 ATT TTG CGT CCA CTT TCA AGC GAT GCT GCA GAT GCT CTA TCAAGA TTC 912 Ile Leu Arg Pro Leu Ser Ser Asp Ala Ala Asp Ala Leu Ser ArgPhe 290 295 300 TAT TAT CCA CCG GTT GCT GCT GTA ACT GTT TCG TAT CCA AAGGAA GCA 960 Tyr Tyr Pro Pro Val Ala Ala Val Thr Val Ser Tyr Pro Lys GluAla 305 310 315 320 ATT AGA AAA GAA TGC TTA ATT GAT GGG GAA CTC CAG GGCTTT GGC CAG 1008 Ile Arg Lys Glu Cys Leu Ile Asp Gly Glu Leu Gln Gly PheGly Gln 325 330 335 TTG CAT CCA CGT AGT CAA GGA GTT GAG ACA TTA GGA ACAATA TAC AGT 1056 Leu His Pro Arg Ser Gln Gly Val Glu Thr Leu Gly Thr IleTyr Ser 340 345 350 TCC TCA CTC TTT CCA AAT CGT GCT CCT GAC GGT AGG GTGTTA CTT CTA 1104 Ser Ser Leu Phe Pro Asn Arg Ala Pro Asp Gly Arg Val LeuLeu Leu 355 360 365 AAC TAC ATA GGA GGT GCT ACA AAC ACA GGA ATT GTT TCCAAG ACT GAA 1152 Asn Tyr Ile Gly Gly Ala Thr Asn Thr Gly Ile Val Ser LysThr Glu 370 375 380 AGT GAG CTG GTC GAA GCA GTT GAC CGT GAC CTC CGA AAAATG CTT ATA 1200 Ser Glu Leu Val Glu Ala Val Asp Arg Asp Leu Arg Lys MetLeu Ile 385 390 395 400 AAT TCT ACA GCA GTG GAC CCT TTA GTC CTT GGT GTTCGA GTT TGG CCA 1248 Asn Ser Thr Ala Val Asp Pro Leu Val Leu Gly Val ArgVal Trp Pro 405 410 415 CAA GCC ATA CCT CAG TTC CTG GTA GGA CAT CTT GATCTT CTG GAA GCC 1296 Gln Ala Ile Pro Gln Phe Leu Val Gly His Leu Asp LeuLeu Glu Ala 420 425 430 GCA AAA GCT GCC CTG GAC CGA GGT GGC TAC GAT GGGCTG TTC CTA GGA 1344 Ala Lys Ala Ala Leu Asp Arg Gly Gly Tyr Asp Gly LeuPhe Leu Gly 435 440 445 GGG AAC TAT GTT GCA GGA GTT GCC CTG GGC AGA TGCGTT GAG GGC GCG 1392 Gly Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys ValGlu Gly Ala 450 455 460 TAT GAA AGT GCC TCG CAA ATA TCT GAC TTC TTG ACCAAG TAT GCC TAC 1440 Tyr Glu Ser Ala Ser Gln Ile Ser Asp Phe Leu Thr LysTyr Ala Tyr 465 470 475 480 AAG TGATGAAAGA AGTGGAGCGC TACTTGTTAATCGTTTATGT TGCATAGATG 1493 Lys AGGTGCCTCC GGGGAAAAAA AAGCTTGAATAGTATTTTTT ATTCTTATTT TGTAAATT 1553 ATTTCTGTTC TTTTTTCTAT CAGTAATTAGTTATATTTTA GTTCTGTAGG AGATTGTT 1613 GTTCACTGCC CTTCAAAAGA AATTTTATTTTTCATTCTTT TATGAGAGCT GTGCTACT 1673 AAAAAAAAAA AAAAAAAA 1691 481 aminoacids amino acid linear protein not provided 6 Ala Asp Cys Val Val ValGly Gly Gly Ile Ser Gly Leu Cys Thr Ala 1 5 10 15 Gln Ala Leu Ala ThrArg His Gly Val Gly Asp Val Leu Val Thr Glu 20 25 30 Ala Arg Ala Arg ProGly Gly Asn Ile Thr Thr Val Glu Arg Pro Glu 35 40 45 Glu Gly Tyr Leu TrpGlu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp 50 55 60 Pro Val Leu Thr MetAla Val Asp Ser Gly Leu Lys Asp Asp Leu Val 65 70 75 80 Phe Gly Asp ProAsn Ala Pro Arg Phe Val Leu Trp Glu Gly Lys Leu 85 90 95 Arg Pro Val ProSer Lys Pro Ala Asp Leu Pro Phe Phe Asp Leu Met 100 105 110 Ser Ile ProGly Lys Leu Arg Ala Gly Leu Gly Ala Leu Gly Ile Arg 115 120 125 Pro ProPro Pro Gly Arg Glu Glu Ser Val Glu Glu Phe Val Arg Arg 130 135 140 AsnLeu Gly Ala Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser 145 150 155160 Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe 165170 175 Gly Lys Val Trp Arg Leu Glu Glu Thr Gly Gly Ser Ile Ile Gly Gly180 185 190 Thr Ile Lys Thr Ile Gln Glu Arg Ser Lys Asn Pro Lys Pro ProArg 195 200 205 Asp Ala Arg Leu Pro Lys Pro Lys Gly Gln Thr Val Ala SerPhe Arg 210 215 220 Lys Gly Leu Ala Met Leu Pro Asn Ala Ile Thr Ser SerLeu Gly Ser 225 230 235 240 Lys Val Lys Leu Ser Trp Lys Leu Thr Ser IleThr Lys Ser Asp Asp 245 250 255 Lys Gly Tyr Val Leu Glu Tyr Glu Thr ProGlu Gly Val Val Ser Val 260 265 270 Gln Ala Lys Ser Val Ile Met Thr IlePro Ser Tyr Val Ala Ser Asn 275 280 285 Ile Leu Arg Pro Leu Ser Ser AspAla Ala Asp Ala Leu Ser Arg Phe 290 295 300 Tyr Tyr Pro Pro Val Ala AlaVal Thr Val Ser Tyr Pro Lys Glu Ala 305 310 315 320 Ile Arg Lys Glu CysLeu Ile Asp Gly Glu Leu Gln Gly Phe Gly Gln 325 330 335 Leu His Pro ArgSer Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr Ser 340 345 350 Ser Ser LeuPhe Pro Asn Arg Ala Pro Asp Gly Arg Val Leu Leu Leu 355 360 365 Asn TyrIle Gly Gly Ala Thr Asn Thr Gly Ile Val Ser Lys Thr Glu 370 375 380 SerGlu Leu Val Glu Ala Val Asp Arg Asp Leu Arg Lys Met Leu Ile 385 390 395400 Asn Ser Thr Ala Val Asp Pro Leu Val Leu Gly Val Arg Val Trp Pro 405410 415 Gln Ala Ile Pro Gln Phe Leu Val Gly His Leu Asp Leu Leu Glu Ala420 425 430 Ala Lys Ala Ala Leu Asp Arg Gly Gly Tyr Asp Gly Leu Phe LeuGly 435 440 445 Gly Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys Val GluGly Ala 450 455 460 Tyr Glu Ser Ala Ser Gln Ile Ser Asp Phe Leu Thr LysTyr Ala Tyr 465 470 475 480 Lys 2061 base pairs nucleic acid singlelinear cDNA NO NO Zea mays (maize) pWDC-3 (NRRL B-21259) CDS 64..1698/product= “Maize protox-2” 7 CTCTCCTACC TCCACCTCCA CGACAACAAG CAAATCCCCATCCAGTTCCA AACCCTAACT 60 CAA ATG CTC GCT TTG ACT GCC TCA GCC TCA TCC GCTTCG TCC CAT CCT 108 Met Leu Ala Leu Thr Ala Ser Ala Ser Ser Ala Ser SerHis Pro 1 5 10 15 TAT CGC CAC GCC TCC GCG CAC ACT CGT CGC CCC CGC CTACGT GCG GTC 156 Tyr Arg His Ala Ser Ala His Thr Arg Arg Pro Arg Leu ArgAla Val 20 25 30 CTC GCG ATG GCG GGC TCC GAC GAC CCC CGT GCA GCG CCC GCCAGA TCG 204 Leu Ala Met Ala Gly Ser Asp Asp Pro Arg Ala Ala Pro Ala ArgSer 35 40 45 GTC GCC GTC GTC GGC GCC GGG GTC AGC GGG CTC GCG GCG GCG TACAGG 252 Val Ala Val Val Gly Ala Gly Val Ser Gly Leu Ala Ala Ala Tyr Arg50 55 60 CTC AGA CAG AGC GGC GTG AAC GTA ACG GTG TTC GAA GCG GCC GAC AGG300 Leu Arg Gln Ser Gly Val Asn Val Thr Val Phe Glu Ala Ala Asp Arg 6570 75 GCG GGA GGA AAG ATA CGG ACC AAT TCC GAG GGC GGG TTT GTC TGG GAT348 Ala Gly Gly Lys Ile Arg Thr Asn Ser Glu Gly Gly Phe Val Trp Asp 8085 90 95 GAA GGA GCT AAC ACC ATG ACA GAA GGT GAA TGG GAG GCC AGT AGA CTG396 Glu Gly Ala Asn Thr Met Thr Glu Gly Glu Trp Glu Ala Ser Arg Leu 100105 110 ATT GAT GAT CTT GGT CTA CAA GAC AAA CAG CAG TAT CCT AAC TCC CAA444 Ile Asp Asp Leu Gly Leu Gln Asp Lys Gln Gln Tyr Pro Asn Ser Gln 115120 125 CAC AAG CGT TAC ATT GTC AAA GAT GGA GCA CCA GCA CTG ATT CCT TCG492 His Lys Arg Tyr Ile Val Lys Asp Gly Ala Pro Ala Leu Ile Pro Ser 130135 140 GAT CCC ATT TCG CTA ATG AAA AGC AGT GTT CTT TCG ACA AAA TCA AAG540 Asp Pro Ile Ser Leu Met Lys Ser Ser Val Leu Ser Thr Lys Ser Lys 145150 155 ATT GCG TTA TTT TTT GAA CCA TTT CTC TAC AAG AAA GCT AAC ACA AGA588 Ile Ala Leu Phe Phe Glu Pro Phe Leu Tyr Lys Lys Ala Asn Thr Arg 160165 170 175 AAC TCT GGA AAA GTG TCT GAG GAG CAC TTG AGT GAG AGT GTT GGGAGC 636 Asn Ser Gly Lys Val Ser Glu Glu His Leu Ser Glu Ser Val Gly Ser180 185 190 TTC TGT GAA CGC CAC TTT GGA AGA GAA GTT GTT GAC TAT TTT GTTGAT 684 Phe Cys Glu Arg His Phe Gly Arg Glu Val Val Asp Tyr Phe Val Asp195 200 205 CCA TTT GTA GCT GGA ACA AGT GCA GGA GAT CCA GAG TCA CTA TCTATT 732 Pro Phe Val Ala Gly Thr Ser Ala Gly Asp Pro Glu Ser Leu Ser Ile210 215 220 CGT CAT GCA TTC CCA GCA TTG TGG AAT TTG GAA AGA AAG TAT GGTTCA 780 Arg His Ala Phe Pro Ala Leu Trp Asn Leu Glu Arg Lys Tyr Gly Ser225 230 235 GTT ATT GTT GGT GCC ATC TTG TCT AAG CTA GCA GCT AAA GGT GATCCA 828 Val Ile Val Gly Ala Ile Leu Ser Lys Leu Ala Ala Lys Gly Asp Pro240 245 250 255 GTA AAG ACA AGA CAT GAT TCA TCA GGG AAA AGA AGG AAT AGACGA GTG 876 Val Lys Thr Arg His Asp Ser Ser Gly Lys Arg Arg Asn Arg ArgVal 260 265 270 TCG TTT TCA TTT CAT GGT GGA ATG CAG TCA CTA ATA AAT GCACTT CAC 924 Ser Phe Ser Phe His Gly Gly Met Gln Ser Leu Ile Asn Ala LeuHis 275 280 285 AAT GAA GTT GGA GAT GAT AAT GTG AAG CTT GGT ACA GAA GTGTTG TCA 972 Asn Glu Val Gly Asp Asp Asn Val Lys Leu Gly Thr Glu Val LeuSer 290 295 300 TTG GCA TGT ACA TTT GAT GGA GTT CCT GCA CTA GGC AGG TGGTCA ATT 1020 Leu Ala Cys Thr Phe Asp Gly Val Pro Ala Leu Gly Arg Trp SerIle 305 310 315 TCT GTT GAT TCG AAG GAT AGC GGT GAC AAG GAC CTT GCT AGTAAC CAA 1068 Ser Val Asp Ser Lys Asp Ser Gly Asp Lys Asp Leu Ala Ser AsnGln 320 325 330 335 ACC TTT GAT GCT GTT ATA ATG ACA GCT CCA TTG TCA AATGTC CGG AGG 1116 Thr Phe Asp Ala Val Ile Met Thr Ala Pro Leu Ser Asn ValArg Arg 340 345 350 ATG AAG TTC ACC AAA GGT GGA GCT CCG GTT GTT CTT GACTTT CTT CCT 1164 Met Lys Phe Thr Lys Gly Gly Ala Pro Val Val Leu Asp PheLeu Pro 355 360 365 AAG ATG GAT TAT CTA CCA CTA TCT CTC ATG GTG ACT GCTTTT AAG AAG 1212 Lys Met Asp Tyr Leu Pro Leu Ser Leu Met Val Thr Ala PheLys Lys 370 375 380 GAT GAT GTC AAG AAA CCT CTG GAA GGA TTT GGG GTC TTAATA CCT TAC 1260 Asp Asp Val Lys Lys Pro Leu Glu Gly Phe Gly Val Leu IlePro Tyr 385 390 395 AAG GAA CAG CAA AAA CAT GGT CTG AAA ACC CTT GGG ACTCTC TTT TCC 1308 Lys Glu Gln Gln Lys His Gly Leu Lys Thr Leu Gly Thr LeuPhe Ser 400 405 410 415 TCA ATG ATG TTC CCA GAT CGA GCT CCT GAT GAC CAATAT TTA TAT ACA 1356 Ser Met Met Phe Pro Asp Arg Ala Pro Asp Asp Gln TyrLeu Tyr Thr 420 425 430 ACA TTT GTT GGG GGT AGC CAC AAT AGA GAT CTT GCTGGA GCT CCA ACG 1404 Thr Phe Val Gly Gly Ser His Asn Arg Asp Leu Ala GlyAla Pro Thr 435 440 445 TCT ATT CTG AAA CAA CTT GTG ACC TCT GAC CTT AAAAAA CTC TTG GGC 1452 Ser Ile Leu Lys Gln Leu Val Thr Ser Asp Leu Lys LysLeu Leu Gly 450 455 460 GTA GAG GGG CAA CCA ACT TTT GTC AAG CAT GTA TACTGG GGA AAT GCT 1500 Val Glu Gly Gln Pro Thr Phe Val Lys His Val Tyr TrpGly Asn Ala 465 470 475 TTT CCT TTG TAT GGC CAT GAT TAT AGT TCT GTA TTGGAA GCT ATA GAA 1548 Phe Pro Leu Tyr Gly His Asp Tyr Ser Ser Val Leu GluAla Ile Glu 480 485 490 495 AAG ATG GAG AAA AAC CTT CCA GGG TTC TTC TACGCA GGA AAT AGC AAG 1596 Lys Met Glu Lys Asn Leu Pro Gly Phe Phe Tyr AlaGly Asn Ser Lys 500 505 510 GAT GGG CTT GCT GTT GGA AGT GTT ATA GCT TCAGGA AGC AAG GCT GCT 1644 Asp Gly Leu Ala Val Gly Ser Val Ile Ala Ser GlySer Lys Ala Ala 515 520 525 GAC CTT GCA ATC TCA TAT CTT GAA TCT CAC ACCAAG CAT AAT AAT TCA 1692 Asp Leu Ala Ile Ser Tyr Leu Glu Ser His Thr LysHis Asn Asn Ser 530 535 540 CAT TGAAAGTGTC TGACCTATCC TCTAGCAGTTGTCGACAAAT TTCTCCAGTT 1745 His 545 CATGTACAGT AGAAACCGAT GCGTTGCAGTTTCAGAACAT CTTCACTTCT TCAGATAT 1805 ACCCTTCGTT GAACATCCAC CAGAAAGGTAGTCACATGTG TAAGTGGGAA AATGAGGT 1865 AAAACTATTA TGGCGGCCGA AATGTTCCTTTTTGTTTTCC TCACAAGTGG CCTACGAC 1925 TTGATGTTGG AAATACATTT AAATTTGTTGAATTGTTTGA GAACACATGC GTGACGTG 1985 ATATTTGCCT ATTGTGATTT TAGCAGTAGTCTTGGCCAGA TTATGCTTTA CGCCTTTA 2045 AAAAAAAAAA AAAAAA 2061 544 aminoacids amino acid linear protein not provided 8 Met Leu Ala Leu Thr AlaSer Ala Ser Ser Ala Ser Ser His Pro Tyr 1 5 10 15 Arg His Ala Ser AlaHis Thr Arg Arg Pro Arg Leu Arg Ala Val Leu 20 25 30 Ala Met Ala Gly SerAsp Asp Pro Arg Ala Ala Pro Ala Arg Ser Val 35 40 45 Ala Val Val Gly AlaGly Val Ser Gly Leu Ala Ala Ala Tyr Arg Leu 50 55 60 Arg Gln Ser Gly ValAsn Val Thr Val Phe Glu Ala Ala Asp Arg Ala 65 70 75 80 Gly Gly Lys IleArg Thr Asn Ser Glu Gly Gly Phe Val Trp Asp Glu 85 90 95 Gly Ala Asn ThrMet Thr Glu Gly Glu Trp Glu Ala Ser Arg Leu Ile 100 105 110 Asp Asp LeuGly Leu Gln Asp Lys Gln Gln Tyr Pro Asn Ser Gln His 115 120 125 Lys ArgTyr Ile Val Lys Asp Gly Ala Pro Ala Leu Ile Pro Ser Asp 130 135 140 ProIle Ser Leu Met Lys Ser Ser Val Leu Ser Thr Lys Ser Lys Ile 145 150 155160 Ala Leu Phe Phe Glu Pro Phe Leu Tyr Lys Lys Ala Asn Thr Arg Asn 165170 175 Ser Gly Lys Val Ser Glu Glu His Leu Ser Glu Ser Val Gly Ser Phe180 185 190 Cys Glu Arg His Phe Gly Arg Glu Val Val Asp Tyr Phe Val AspPro 195 200 205 Phe Val Ala Gly Thr Ser Ala Gly Asp Pro Glu Ser Leu SerIle Arg 210 215 220 His Ala Phe Pro Ala Leu Trp Asn Leu Glu Arg Lys TyrGly Ser Val 225 230 235 240 Ile Val Gly Ala Ile Leu Ser Lys Leu Ala AlaLys Gly Asp Pro Val 245 250 255 Lys Thr Arg His Asp Ser Ser Gly Lys ArgArg Asn Arg Arg Val Ser 260 265 270 Phe Ser Phe His Gly Gly Met Gln SerLeu Ile Asn Ala Leu His Asn 275 280 285 Glu Val Gly Asp Asp Asn Val LysLeu Gly Thr Glu Val Leu Ser Leu 290 295 300 Ala Cys Thr Phe Asp Gly ValPro Ala Leu Gly Arg Trp Ser Ile Ser 305 310 315 320 Val Asp Ser Lys AspSer Gly Asp Lys Asp Leu Ala Ser Asn Gln Thr 325 330 335 Phe Asp Ala ValIle Met Thr Ala Pro Leu Ser Asn Val Arg Arg Met 340 345 350 Lys Phe ThrLys Gly Gly Ala Pro Val Val Leu Asp Phe Leu Pro Lys 355 360 365 Met AspTyr Leu Pro Leu Ser Leu Met Val Thr Ala Phe Lys Lys Asp 370 375 380 AspVal Lys Lys Pro Leu Glu Gly Phe Gly Val Leu Ile Pro Tyr Lys 385 390 395400 Glu Gln Gln Lys His Gly Leu Lys Thr Leu Gly Thr Leu Phe Ser Ser 405410 415 Met Met Phe Pro Asp Arg Ala Pro Asp Asp Gln Tyr Leu Tyr Thr Thr420 425 430 Phe Val Gly Gly Ser His Asn Arg Asp Leu Ala Gly Ala Pro ThrSer 435 440 445 Ile Leu Lys Gln Leu Val Thr Ser Asp Leu Lys Lys Leu LeuGly Val 450 455 460 Glu Gly Gln Pro Thr Phe Val Lys His Val Tyr Trp GlyAsn Ala Phe 465 470 475 480 Pro Leu Tyr Gly His Asp Tyr Ser Ser Val LeuGlu Ala Ile Glu Lys 485 490 495 Met Glu Lys Asn Leu Pro Gly Phe Phe TyrAla Gly Asn Ser Lys Asp 500 505 510 Gly Leu Ala Val Gly Ser Val Ile AlaSer Gly Ser Lys Ala Ala Asp 515 520 525 Leu Ala Ile Ser Tyr Leu Glu SerHis Thr Lys His Asn Asn Ser His 530 535 540 1811 base pairs nucleic acidsingle linear cDNA NO Triticum aestivum (wheat) pWDC-13 (NRRL B-21545)CDS 3..1589 /product= “wheat protox-1” 9 GC GCA ACA ATG GCC ACC GCC ACCGTC GCG GCC GCG TCG CCG CTC CGC 47 Ala Thr Met Ala Thr Ala Thr Val AlaAla Ala Ser Pro Leu Arg 1 5 10 15 GGC AGG GTC ACC GGG CGC CCA CAC CGCGTC CGC CCG CGT TGC GCT ACC 95 Gly Arg Val Thr Gly Arg Pro His Arg ValArg Pro Arg Cys Ala Thr 20 25 30 GCG AGC AGC GCG ACC GAG ACT CCG GCG GCGCCC GGC GTG CGG CTG TCC 143 Ala Ser Ser Ala Thr Glu Thr Pro Ala Ala ProGly Val Arg Leu Ser 35 40 45 GCG GAA TGC GTC ATT GTG GGC GCC GGC ATC AGCGGC CTC TGC ACC GCG 191 Ala Glu Cys Val Ile Val Gly Ala Gly Ile Ser GlyLeu Cys Thr Ala 50 55 60 CAG GCG CTG GCC ACC CGA TAC GGC GTC AGC GAC CTGCTC GTC ACG GAG 239 Gln Ala Leu Ala Thr Arg Tyr Gly Val Ser Asp Leu LeuVal Thr Glu 65 70 75 GCC CGC GAC CGC CCG GGC GGC AAC ATC ACC ACC GTC GAGCGT CCC GAC 287 Ala Arg Asp Arg Pro Gly Gly Asn Ile Thr Thr Val Glu ArgPro Asp 80 85 90 95 GAG GGG TAC CTG TGG GAG GAG GGA CCC AAC AGC TTC CAGCCC TCC GAC 335 Glu Gly Tyr Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln ProSer Asp 100 105 110 CCG GTC CTC ACC ATG GCC GTG GAC AGC GGG CTC AAG GATGAC TTG GTG 383 Pro Val Leu Thr Met Ala Val Asp Ser Gly Leu Lys Asp AspLeu Val 115 120 125 TTC GGG GAC CCC AAC GCG CCC CGG TTC GTG CTG TGG GAGGGG AAG CTG 431 Phe Gly Asp Pro Asn Ala Pro Arg Phe Val Leu Trp Glu GlyLys Leu 130 135 140 AGG CCG GTG CCG TCG AAG CCA GGC GAC CTG CCT TTC TTCAGC CTC ATG 479 Arg Pro Val Pro Ser Lys Pro Gly Asp Leu Pro Phe Phe SerLeu Met 145 150 155 AGT ATC CCT GGG AAG CTC AGG GCC GGC CTT GGC GCG CTCGGC ATT CGC 527 Ser Ile Pro Gly Lys Leu Arg Ala Gly Leu Gly Ala Leu GlyIle Arg 160 165 170 175 CCA CCT CCT CCA GGG CGC GAG GAG TCG GTG GAG GAGTTT GTG CGC CGC 575 Pro Pro Pro Pro Gly Arg Glu Glu Ser Val Glu Glu PheVal Arg Arg 180 185 190 AAC CTC GGT GCC GAG GTC TTT GAG CGC CTC ATC GAGCCT TTC TGC TCA 623 Asn Leu Gly Ala Glu Val Phe Glu Arg Leu Ile Glu ProPhe Cys Ser 195 200 205 GGT GTA TAT GCT GGT GAT CCT TCG AAG CTT AGT ATGAAG GCT GCA TTT 671 Gly Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met LysAla Ala Phe 210 215 220 GGG AAG GTC TGG AGG TTG GAG GAG ATT GGA GGT AGTATT ATT GGT GGA 719 Gly Lys Val Trp Arg Leu Glu Glu Ile Gly Gly Ser IleIle Gly Gly 225 230 235 ACC ATC AAG GCG ATT CAG GAT AAA GGG AAG AAC CCCAAA CCG CCA AGG 767 Thr Ile Lys Ala Ile Gln Asp Lys Gly Lys Asn Pro LysPro Pro Arg 240 245 250 255 GAT CCC CGA CTT CCG GCA CCA AAG GGA CAG ACGGTG GCA TCT TTC AGG 815 Asp Pro Arg Leu Pro Ala Pro Lys Gly Gln Thr ValAla Ser Phe Arg 260 265 270 AAG GGT CTA GCC ATG CTC CCG AAT GCC ATC GCATCT AGG CTG GGT AGT 863 Lys Gly Leu Ala Met Leu Pro Asn Ala Ile Ala SerArg Leu Gly Ser 275 280 285 AAA GTC AAG CTG TCA TGG AAG CTT ACG AGC ATTACA AAG GCG GAC AAC 911 Lys Val Lys Leu Ser Trp Lys Leu Thr Ser Ile ThrLys Ala Asp Asn 290 295 300 CAA GGA TAT GTA TTA GGT TAT GAA ACA CCA GAAGGA CTT GTT TCA GTG 959 Gln Gly Tyr Val Leu Gly Tyr Glu Thr Pro Glu GlyLeu Val Ser Val 305 310 315 CAG GCT AAA AGT GTT ATC ATG ACC ATC CCG TCATAT GTT GCT AGT GAT 1007 Gln Ala Lys Ser Val Ile Met Thr Ile Pro Ser TyrVal Ala Ser Asp 320 325 330 335 ATC TTG CGC CCA CTT TCA ATT GAT GCA GCAGAT GCA CTC TCA AAA TTC 1055 Ile Leu Arg Pro Leu Ser Ile Asp Ala Ala AspAla Leu Ser Lys Phe 340 345 350 TAT TAT CCG CCA GTT GCT GCT GTA ACT GTTTCA TAT CCA AAA GAA GCT 1103 Tyr Tyr Pro Pro Val Ala Ala Val Thr Val SerTyr Pro Lys Glu Ala 355 360 365 ATT AGA AAA GAA TGC TTA ATT GAT GGG GAGCTC CAG GGT TTC GGC CAG 1151 Ile Arg Lys Glu Cys Leu Ile Asp Gly Glu LeuGln Gly Phe Gly Gln 370 375 380 TTG CAT CCA CGT AGC CAA GGA GTC GAG ACTTTA GGG ACA ATA TAT AGC 1199 Leu His Pro Arg Ser Gln Gly Val Glu Thr LeuGly Thr Ile Tyr Ser 385 390 395 TCT TCT CTC TTT CCT AAT CGT GCT CCT GCTGGA AGA GTG TTA CTT CTG 1247 Ser Ser Leu Phe Pro Asn Arg Ala Pro Ala GlyArg Val Leu Leu Leu 400 405 410 415 AAC TAT ATC GGG GGT TCT ACA AAT ACAGGG ATC GTC TCC AAG ACT GAG 1295 Asn Tyr Ile Gly Gly Ser Thr Asn Thr GlyIle Val Ser Lys Thr Glu 420 425 430 AGT GAC TTA GTA GGA GCC GTT GAC CGTGAC CTC AGA AAA ATG TTG ATA 1343 Ser Asp Leu Val Gly Ala Val Asp Arg AspLeu Arg Lys Met Leu Ile 435 440 445 AAC CCT AGA GCA GCA GAC CCT TTA GCATTA GGG GTT CGA GTG TGG CCA 1391 Asn Pro Arg Ala Ala Asp Pro Leu Ala LeuGly Val Arg Val Trp Pro 450 455 460 CAA GCA ATA CCA CAG TTT TTG ATT GGGCAC CTT GAT CGC CTT GCT GCT 1439 Gln Ala Ile Pro Gln Phe Leu Ile Gly HisLeu Asp Arg Leu Ala Ala 465 470 475 GCA AAA TCT GCA CTG GGC CAA GGC GGCTAC GAC GGG TTG TTC CTA GGA 1487 Ala Lys Ser Ala Leu Gly Gln Gly Gly TyrAsp Gly Leu Phe Leu Gly 480 485 490 495 GGA AAC TAC GTC GCA GGA GTT GCCTTG GGC CGA TGC ATC GAG GGT GCG 1535 Gly Asn Tyr Val Ala Gly Val Ala LeuGly Arg Cys Ile Glu Gly Ala 500 505 510 TAC GAG AGT GCC TCA CAA GTA TCTGAC TTC TTG ACC AAG TAT GCC TAC 1583 Tyr Glu Ser Ala Ser Gln Val Ser AspPhe Leu Thr Lys Tyr Ala Tyr 515 520 525 AAG TGA TGGAAGTAGT GCATCTCTTCATTTTGTTGC ATATACGAGG TGAGGCTAGG 1639 Lys ATCGGTAAAA CATCATGAGATTCTGTAGTG TTTCTTTAAT TGAAAAAACA AATTTTAG 1699 ATGCAATATG TGCTCTTTCCTGTAGTTCGA GCATGTACAT CGGTATGGGA TAAAGTAG 1759 TAAGCTATTC TGCAAAAGCAGTGATTTTTT TTGAAAAAAA AAAAAAAAAA AA 1811 528 amino acids amino acidlinear protein not provided 10 Ala Thr Met Ala Thr Ala Thr Val Ala AlaAla Ser Pro Leu Arg Gly 1 5 10 15 Arg Val Thr Gly Arg Pro His Arg ValArg Pro Arg Cys Ala Thr Ala 20 25 30 Ser Ser Ala Thr Glu Thr Pro Ala AlaPro Gly Val Arg Leu Ser Ala 35 40 45 Glu Cys Val Ile Val Gly Ala Gly IleSer Gly Leu Cys Thr Ala Gln 50 55 60 Ala Leu Ala Thr Arg Tyr Gly Val SerAsp Leu Leu Val Thr Glu Ala 65 70 75 80 Arg Asp Arg Pro Gly Gly Asn IleThr Thr Val Glu Arg Pro Asp Glu 85 90 95 Gly Tyr Leu Trp Glu Glu Gly ProAsn Ser Phe Gln Pro Ser Asp Pro 100 105 110 Val Leu Thr Met Ala Val AspSer Gly Leu Lys Asp Asp Leu Val Phe 115 120 125 Gly Asp Pro Asn Ala ProArg Phe Val Leu Trp Glu Gly Lys Leu Arg 130 135 140 Pro Val Pro Ser LysPro Gly Asp Leu Pro Phe Phe Ser Leu Met Ser 145 150 155 160 Ile Pro GlyLys Leu Arg Ala Gly Leu Gly Ala Leu Gly Ile Arg Pro 165 170 175 Pro ProPro Gly Arg Glu Glu Ser Val Glu Glu Phe Val Arg Arg Asn 180 185 190 LeuGly Ala Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser Gly 195 200 205Val Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe Gly 210 215220 Lys Val Trp Arg Leu Glu Glu Ile Gly Gly Ser Ile Ile Gly Gly Thr 225230 235 240 Ile Lys Ala Ile Gln Asp Lys Gly Lys Asn Pro Lys Pro Pro ArgAsp 245 250 255 Pro Arg Leu Pro Ala Pro Lys Gly Gln Thr Val Ala Ser PheArg Lys 260 265 270 Gly Leu Ala Met Leu Pro Asn Ala Ile Ala Ser Arg LeuGly Ser Lys 275 280 285 Val Lys Leu Ser Trp Lys Leu Thr Ser Ile Thr LysAla Asp Asn Gln 290 295 300 Gly Tyr Val Leu Gly Tyr Glu Thr Pro Glu GlyLeu Val Ser Val Gln 305 310 315 320 Ala Lys Ser Val Ile Met Thr Ile ProSer Tyr Val Ala Ser Asp Ile 325 330 335 Leu Arg Pro Leu Ser Ile Asp AlaAla Asp Ala Leu Ser Lys Phe Tyr 340 345 350 Tyr Pro Pro Val Ala Ala ValThr Val Ser Tyr Pro Lys Glu Ala Ile 355 360 365 Arg Lys Glu Cys Leu IleAsp Gly Glu Leu Gln Gly Phe Gly Gln Leu 370 375 380 His Pro Arg Ser GlnGly Val Glu Thr Leu Gly Thr Ile Tyr Ser Ser 385 390 395 400 Ser Leu PhePro Asn Arg Ala Pro Ala Gly Arg Val Leu Leu Leu Asn 405 410 415 Tyr IleGly Gly Ser Thr Asn Thr Gly Ile Val Ser Lys Thr Glu Ser 420 425 430 AspLeu Val Gly Ala Val Asp Arg Asp Leu Arg Lys Met Leu Ile Asn 435 440 445Pro Arg Ala Ala Asp Pro Leu Ala Leu Gly Val Arg Val Trp Pro Gln 450 455460 Ala Ile Pro Gln Phe Leu Ile Gly His Leu Asp Arg Leu Ala Ala Ala 465470 475 480 Lys Ser Ala Leu Gly Gln Gly Gly Tyr Asp Gly Leu Phe Leu GlyGly 485 490 495 Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys Ile Glu GlyAla Tyr 500 505 510 Glu Ser Ala Ser Gln Val Ser Asp Phe Leu Thr Lys TyrAla Tyr Lys 515 520 525 1847 base pairs nucleic acid single linear cDNANO soybean pWDC-12 (NRRL B-21516) CDS 55..1683 /product= “soybeanprotox-1” 11 CTTTAGCACA GTGTTGAAGA TAACGAACGA ATAGTGCCAT TACTGTAACC AACCATG 57 Met 1 GTT TCC GTC TTC AAC GAG ATC CTA TTC CCG CCG AAC CAA ACC CTTCTT 105 Val Ser Val Phe Asn Glu Ile Leu Phe Pro Pro Asn Gln Thr Leu Leu5 10 15 CGC CCC TCC CTC CAT TCC CCA ACC TCT TTC TTC ACC TCT CCC ACT CGA153 Arg Pro Ser Leu His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr Arg 2025 30 AAA TTC CCT CGC TCT CGC CCT AAC CCT ATT CTA CGC TGC TCC ATT GCG201 Lys Phe Pro Arg Ser Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile Ala 3540 45 GAG GAA TCC ACC GCG TCT CCG CCC AAA ACC AGA GAC TCC GCC CCC GTG249 Glu Glu Ser Thr Ala Ser Pro Pro Lys Thr Arg Asp Ser Ala Pro Val 5055 60 65 GAC TGC GTC GTC GTC GGC GGA GGC GTC AGC GGC CTC TGC ATC GCC CAG297 Asp Cys Val Val Val Gly Gly Gly Val Ser Gly Leu Cys Ile Ala Gln 7075 80 GCC CTC GCC ACC AAA CAC GCC AAT GCC AAC GTC GTC GTC ACG GAG GCC345 Ala Leu Ala Thr Lys His Ala Asn Ala Asn Val Val Val Thr Glu Ala 8590 95 CGA GAC CGC GTC GGC GGC AAC ATC ACC ACG ATG GAG AGG GAC GGA TAC393 Arg Asp Arg Val Gly Gly Asn Ile Thr Thr Met Glu Arg Asp Gly Tyr 100105 110 CTC TGG GAA GAA GGC CCC AAC AGC TTC CAG CCT TCT GAT CCA ATG CTC441 Leu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp Pro Met Leu 115120 125 ACC ATG GTG GTG GAC AGT GGT TTA AAG GAT GAG CTT GTT TTG GGG GAT489 Thr Met Val Val Asp Ser Gly Leu Lys Asp Glu Leu Val Leu Gly Asp 130135 140 145 CCT GAT GCA CCT CGG TTT GTG TTG TGG AAC AGG AAG TTG AGG CCGGTG 537 Pro Asp Ala Pro Arg Phe Val Leu Trp Asn Arg Lys Leu Arg Pro Val150 155 160 CCC GGG AAG CTG ACT GAT TTG CCT TTC TTT GAC TTG ATG AGC ATTGGT 585 Pro Gly Lys Leu Thr Asp Leu Pro Phe Phe Asp Leu Met Ser Ile Gly165 170 175 GGC AAA ATC AGG GCT GGC TTT GGT GCG CTT GGA ATT CGG CCT CCTCCT 633 Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu Gly Ile Arg Pro Pro Pro180 185 190 CCA GGT CAT GAG GAA TCG GTT GAA GAG TTT GTT CGT CGG AAC CTTGGT 681 Pro Gly His Glu Glu Ser Val Glu Glu Phe Val Arg Arg Asn Leu Gly195 200 205 GAT GAG GTT TTT GAA CGG TTG ATA GAG CCT TTT TGT TCA GGG GTCTAT 729 Asp Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr210 215 220 225 GCA GGC GAT CCT TCA AAA TTA AGT ATG AAA GCA GCA TTC GGGAAA GTT 777 Ala Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe Gly LysVal 230 235 240 TGG AAG CTG GAA AAA AAT GGT GGT AGC ATT ATT GGT GGA ACTTTC AAA 825 Trp Lys Leu Glu Lys Asn Gly Gly Ser Ile Ile Gly Gly Thr PheLys 245 250 255 GCA ATA CAA GAG AGA AAT GGA GCT TCA AAA CCA CCT CGA GATCCG CGT 873 Ala Ile Gln Glu Arg Asn Gly Ala Ser Lys Pro Pro Arg Asp ProArg 260 265 270 CTG CCA AAA CCA AAA GGT CAG ACT GTT GGA TCT TTC CGG AAGGGA CTT 921 Leu Pro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe Arg Lys GlyLeu 275 280 285 ACC ATG TTG CCT GAT GCA ATT TCT GCC AGA CTA GGC AAC AAAGTA AAG 969 Thr Met Leu Pro Asp Ala Ile Ser Ala Arg Leu Gly Asn Lys ValLys 290 295 300 305 TTA TCT TGG AAG CTT TCA AGT ATT AGT AAA CTG GAT AGTGGA GAG TAC 1017 Leu Ser Trp Lys Leu Ser Ser Ile Ser Lys Leu Asp Ser GlyGlu Tyr 310 315 320 AGT TTG ACA TAT GAA ACA CCA GAA GGA GTG GTT TCT TTGCAG TGC AAA 1065 Ser Leu Thr Tyr Glu Thr Pro Glu Gly Val Val Ser Leu GlnCys Lys 325 330 335 ACT GTT GTC CTG ACC ATT CCT TCC TAT GTT GCT AGT ACATTG CTG CGT 1113 Thr Val Val Leu Thr Ile Pro Ser Tyr Val Ala Ser Thr LeuLeu Arg 340 345 350 CCT CTG TCT GCT GCT GCT GCA GAT GCA CTT TCA AAG TTTTAT TAC CCT 1161 Pro Leu Ser Ala Ala Ala Ala Asp Ala Leu Ser Lys Phe TyrTyr Pro 355 360 365 CCA GTT GCT GCA GTT TCC ATA TCC TAT CCA AAA GAA GCTATT AGA TCA 1209 Pro Val Ala Ala Val Ser Ile Ser Tyr Pro Lys Glu Ala IleArg Ser 370 375 380 385 GAA TGC TTG ATA GAT GGT GAG TTG AAG GGG TTT GGTCAA TTG CAT CCA 1257 Glu Cys Leu Ile Asp Gly Glu Leu Lys Gly Phe Gly GlnLeu His Pro 390 395 400 CGT AGC CAA GGA GTG GAA ACA TTA GGA ACT ATA TACAGC TCA TCA CTA 1305 Arg Ser Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr SerSer Ser Leu 405 410 415 TTC CCC AAC CGA GCA CCA CCT GGA AGG GTT CTA CTCTTG AAT TAC ATT 1353 Phe Pro Asn Arg Ala Pro Pro Gly Arg Val Leu Leu LeuAsn Tyr Ile 420 425 430 GGA GGA GCA ACT AAT ACT GGA ATT TTA TCG AAG ACGGAC AGT GAA CTT 1401 Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys Thr AspSer Glu Leu 435 440 445 GTG GAA ACA GTT GAT CGA GAT TTG AGG AAA ATC CTTATA AAC CCA AAT 1449 Val Glu Thr Val Asp Arg Asp Leu Arg Lys Ile Leu IleAsn Pro Asn 450 455 460 465 GCC CAG GAT CCA TTT GTA GTG GGG GTG AGA CTGTGG CCT CAA GCT ATT 1497 Ala Gln Asp Pro Phe Val Val Gly Val Arg Leu TrpPro Gln Ala Ile 470 475 480 CCA CAG TTC TTA GTT GGC CAT CTT GAT CTT CTAGAT GTT GCT AAA GCT 1545 Pro Gln Phe Leu Val Gly His Leu Asp Leu Leu AspVal Ala Lys Ala 485 490 495 TCT ATC AGA AAT ACT GGG TTT GAA GGG CTC TTCCTT GGG GGT AAT TAT 1593 Ser Ile Arg Asn Thr Gly Phe Glu Gly Leu Phe LeuGly Gly Asn Tyr 500 505 510 GTG TCT GGT GTT GCC TTG GGA CGA TGC GTT GAGGGA GCC TAT GAG GTA 1641 Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu GlyAla Tyr Glu Val 515 520 525 GCA GCT GAA GTA AAC GAT TTT CTC ACA AAT AGAGTG TAC AAA 1683 Ala Ala Glu Val Asn Asp Phe Leu Thr Asn Arg Val Tyr Lys530 535 540 TAGTAGCAGT TTTTGTTTTT GTGGTGGAAT GGGTGATGGG ACTCTCGTGTTCCATTGA 1743 TATAATAATG TGAAAGTTTC TCAAATTCGT TCGATAGGTT TTTGGCGGCTTCTATTGC 1803 ATAATGTAAA ATCCTCTTTA AGTTTGAAAA AAAAAAAAAA AAAA 1847 543amino acids amino acid linear protein not provided 12 Met Val Ser ValPhe Asn Glu Ile Leu Phe Pro Pro Asn Gln Thr Leu 1 5 10 15 Leu Arg ProSer Leu His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr 20 25 30 Arg Lys PhePro Arg Ser Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile 35 40 45 Ala Glu GluSer Thr Ala Ser Pro Pro Lys Thr Arg Asp Ser Ala Pro 50 55 60 Val Asp CysVal Val Val Gly Gly Gly Val Ser Gly Leu Cys Ile Ala 65 70 75 80 Gln AlaLeu Ala Thr Lys His Ala Asn Ala Asn Val Val Val Thr Glu 85 90 95 Ala ArgAsp Arg Val Gly Gly Asn Ile Thr Thr Met Glu Arg Asp Gly 100 105 110 TyrLeu Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser Asp Pro Met 115 120 125Leu Thr Met Val Val Asp Ser Gly Leu Lys Asp Glu Leu Val Leu Gly 130 135140 Asp Pro Asp Ala Pro Arg Phe Val Leu Trp Asn Arg Lys Leu Arg Pro 145150 155 160 Val Pro Gly Lys Leu Thr Asp Leu Pro Phe Phe Asp Leu Met SerIle 165 170 175 Gly Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu Gly Ile ArgPro Pro 180 185 190 Pro Pro Gly His Glu Glu Ser Val Glu Glu Phe Val ArgArg Asn Leu 195 200 205 Gly Asp Glu Val Phe Glu Arg Leu Ile Glu Pro PheCys Ser Gly Val 210 215 220 Tyr Ala Gly Asp Pro Ser Lys Leu Ser Met LysAla Ala Phe Gly Lys 225 230 235 240 Val Trp Lys Leu Glu Lys Asn Gly GlySer Ile Ile Gly Gly Thr Phe 245 250 255 Lys Ala Ile Gln Glu Arg Asn GlyAla Ser Lys Pro Pro Arg Asp Pro 260 265 270 Arg Leu Pro Lys Pro Lys GlyGln Thr Val Gly Ser Phe Arg Lys Gly 275 280 285 Leu Thr Met Leu Pro AspAla Ile Ser Ala Arg Leu Gly Asn Lys Val 290 295 300 Lys Leu Ser Trp LysLeu Ser Ser Ile Ser Lys Leu Asp Ser Gly Glu 305 310 315 320 Tyr Ser LeuThr Tyr Glu Thr Pro Glu Gly Val Val Ser Leu Gln Cys 325 330 335 Lys ThrVal Val Leu Thr Ile Pro Ser Tyr Val Ala Ser Thr Leu Leu 340 345 350 ArgPro Leu Ser Ala Ala Ala Ala Asp Ala Leu Ser Lys Phe Tyr Tyr 355 360 365Pro Pro Val Ala Ala Val Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg 370 375380 Ser Glu Cys Leu Ile Asp Gly Glu Leu Lys Gly Phe Gly Gln Leu His 385390 395 400 Pro Arg Ser Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr Ser SerSer 405 410 415 Leu Phe Pro Asn Arg Ala Pro Pro Gly Arg Val Leu Leu LeuAsn Tyr 420 425 430 Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys ThrAsp Ser Glu 435 440 445 Leu Val Glu Thr Val Asp Arg Asp Leu Arg Lys IleLeu Ile Asn Pro 450 455 460 Asn Ala Gln Asp Pro Phe Val Val Gly Val ArgLeu Trp Pro Gln Ala 465 470 475 480 Ile Pro Gln Phe Leu Val Gly His LeuAsp Leu Leu Asp Val Ala Lys 485 490 495 Ala Ser Ile Arg Asn Thr Gly PheGlu Gly Leu Phe Leu Gly Gly Asn 500 505 510 Tyr Val Ser Gly Val Ala LeuGly Arg Cys Val Glu Gly Ala Tyr Glu 515 520 525 Val Ala Ala Glu Val AsnAsp Phe Leu Thr Asn Arg Val Tyr Lys 530 535 540 583 base pairs nucleicacid single linear DNA (genomic) NO not provided promoter 1..583/function= “arabidopsis protox-1 promoter” 13 GAATTCCGAT CGAATTATATAATTATCATA AATTTGAATA AGCATGTTGC CTTTTATTAA 60 AGAGGTTTAA TAAAGTTTGGTAATAATGGA CTTTGACTTC AAACTCGATT CTCATGTAAT 120 TAATTAATAT TTACATCAAAATTTGGTCAC TAATATTACC AAATTAATAT ACTAAAATGT 180 TAATTCGCAA ATAAAACACTAATTCCAAAT AAAGGGTCAT TATGATAAAC ACGTATTGAA 240 CTTGATAAAG CAAAGCAAAAATAATGGGTT TCAAGGTTTG GGTTATATAT GACAAAAAAA 300 AAAAAAGGTT TGGTTATATATCTATTGGGC CTATAACCAT GTTATACAAA TTTGGGCCTA 360 ACTAAAATAA TAAAATAAACGTAATGGTCC TTTTTATATT TGGGTCAAAC CCAACTCTAA 420 ACCCAAACCA AAGAAAAAGTATACGGTACG GTACACAGAC TTATGGTGTG TGTGATTGCA 480 GGTGAATATT TCTCGTCGTCTTCTCCTTTC TTCTGAAGAA GATTACCCAA TCTGAAAAAA 540 ACCAAGAAGC TGACAAAATTCCGAATTCTC TGCGATTTCC ATG 583 3848 base pairs nucleic acid single linearDNA (genomic) NO not provided promoter 1..3848 /function= “maizeprotox-1 promoter” 14 TCGATCTTTC TAGGCTGATC CCCAAATCTT CCTCCGAAGCCCCTGGCGCC TCTGCCCCTT 60 GGAGCTGGTG GCCTGAAAGA GCTTTGCTGT TGCCCCGAAGATTGTGAGGT ATATTGTGAC 120 CTCTGAGACT GACTTCCTTT GTCGTCACTT TGAGTGGAGTTATGGATTGA CCTGACGTGC 180 CTCAGATGGA TTCTTCCTCC GAAGCCCCTG GTCATTTCGGAGAATCTGTA ATCTTATTCC 240 CTTCTTTGGC GAAAATCTGT CAGCTTGGAT GTACTCATCCATCTTCTGAA GCAGCTTCTC 300 CAGAGTTTGT GGAGGCTTCC TGGCGAAATA TTGGGCTGTAGGTCCTGGAC GAAGACCCTT 360 GATCATGGCC TCAATGACAA TCTCATTGGG CACCGTAGGCGCTTGTGCCC TCAATCGCAA 420 GAACCTTCGT ACATATGCCT GAAGGTATTC TTCGTGATCTTGTGTGCATT GGAACAGAGC 480 CTGAGCTGTG ACCGACTTCG TTTGAAAGCC TTGGAAGCTAGTAACCAACA TGTGCTTAAG 540 CTTCTGCCAC GACGTGATAG TCCCTGGCCG AAGAGAAGAATACCATGTTT GGGCTACATT 600 CCGGACTGCC ATGACGAAGG ACTTCGCCAT GACTACAGTGTTGACCCCAT ACGAAGATAT 660 AGTTGCTTCG TAGCTCATCA GAAACTGCTT TGGATCTGAGTGCCCATCAT ACATGGGGAG 720 CTGAGGTGGC TTGTATGATG GGGGCCATGG GGTAGCCTGCAGTTCTGCTG CCAAGGGAGA 780 AGCATCATCA AAAGTAAAGG CATCATGATT AAAATCATCATACCATCCAT CCTCGTTGAA 840 TAAGCCTTCT TGACGAAGCT CCCTGTGTTG GGGCCTTCGATCTTGTTCAT CTTGAACAAG 900 ATGACGCACT TCTTCAGTGG CTTCGTCGAT CTTTCTTTGGAGATCAGCCA GTCGCACCAT 960 CTTCTCCTTC TTTCTTTGTA CTTGTTGATG GATGATCTCCATGTCCCTGA TCTCTTGGTC 1020 CAACTCCTCC TCTTGGAGTG TCAGACTGGT GGCTTTCCTCTTCTGGCTTC GAGCCTCTCG 1080 AAGAGAAAGA GTTTCTTGAT TTGGGTCCAG CGGCTGCAGTGCAGTGGTCC CTGGTGCTGA 1140 AGCTTTCTTC GGTGGCATGA CAAAGGTCAG TGCTTGCCGAAGGTGGTCGA AAAGGGTTCA 1200 CTAGAGGTGG GAGCCAATGT TGGGGACTTC TCAAGTGCTATGAGTTAAGA ACAAGGCAAC 1260 ACAAAATGTT AAATATTAAT AGCTTTCATC TTTCGAAGCATTATTTCCCT TTGGGTATAA 1320 TGATCTTCAG ACGAAAGAGT CCTTCATCAT TGCGATATATGTTAATAGAA GGAGGAGCAT 1380 ATGAAATGTA AGAGACAACA TGAACAATCG TGTAGCATTGTTAATTCATC ATCATTTTAT 1440 TATTATGGAA AAATAGAAAC AATATTGAAT TACAAATGTACCTTTGGCTT GACAGAAGAT 1500 AAAAGTACAA GCTTGACGCA CGAGCAAGTA CAAGTCAGTGTGAACAGTAC GGGGGTACTG 1560 TTCATCTATT TATAGGCACA GGACACAGCC TGTGAGAAATTACAGTCATG CCCTTTACAT 1620 TTACTATTGA CTTATAGAAA AATCTATGAG GACTGGATAGCCTTTTCCCC TTTAAGTCGG 1680 TGCCTTTTTC CGCGATTAAG CCGAATCTCC CTTGCGCATAGCTTCGGAGC ATCGGCAACC 1740 TTCGTCACGA TCATGCCCTT CTCATTGTGT ATGCTTTTAATCCTGAATTC GAAGGTACCT 1800 GTCCATAAAC CATACTTGGA AGACATTGTT AAATTATGTTTTTGAGGACC TTCGGAGGAC 1860 GAAGGCCCCC AACAGTCGTG TTTTTGAGGA CCTTCGGAAGATGAAGGCCC CCAACAAGAC 1920 CTATCCATAA AACCAACCTA TCCACAAAAC CGACCCCATTCACCCTTCAT TTGCCTCACC 1980 AACAACCCTA ATTAGGTTGT TGGTTTAAAT TTTTTAGGGTCAATTTGGTC ATCACCATCC 2040 ACTGTCACTC CACAAACTCA ATATCAATAA ACAGACTCAATCACCCAAAC TGACCATACC 2100 CATAAAACCG CCCCACCCTT CTAGCGCCTC GCCAGAAACCAGAAACCCTG ATTCAGAGTT 2160 CAAACTTAAA ACGACCATAA CTTTCACCTT GGAACTCGAATCAGGTCCAT TTTTTTCCAA 2220 ATCACACAAA ATTAAATTTC GCATCCGATA ATCAAGCCATCTCTTCACTA TGGTTTTAAG 2280 TGTTGCTCAC ACTAGTGTAT TTATGGACTA ATCACCTGTGTATCTCATAC AATAACATAT 2340 CAGTACATCT AAGTTGTTAC TCAATTACCA AAACCGAATTATAGCCTTCG AAAAAGGTTA 2400 TCGACTAGTC ACTCAATTAC CAAAACTAAA CTTTAGACTTTCATGTATGA CATCCAACAT 2460 GACACTGTAC TGGACTAAAC CACCTTTCAA GCTACACAAGGAGCAAAAAT AACTAATTTT 2520 CGTAGTTGTA GGAGCTAAAG TATATGTCCA CAACAATAGTTAAGGGAAGC CCCCAAGGAC 2580 TTAAAAGTCC TTTTACCTCT TGAAACTTTT GTCGTGGTCTACTTTTTCAC TTTAAACTTC 2640 AAAATTTGAC ATTTTATCAC CCCTTAACTC TTAAAACCATTTAAATTACA TTCTTACTAG 2700 ATTATAGATG ATTTTGTTGT GAAAAGTTTT TAAGACATGTTTACACATTG ATTAAAATCA 2760 TTTGTTCAAT TTCCTAGAGT TAAATCTAAT CTTATTAAAACTATTAGAGA TACTTTCACG 2820 AGCTCTAAAT ATTTTTATTT TTTCATTATG GAATTTTGTTAGAATTCTTA TAGACCTTTT 2880 TTTGTGGTTT AAAAGCCTTG CCATGTTTTT AACAAGTTTTTTTTCTATTT TTTGAAATTT 2940 TCTTGGAAAC CACTTCTAAC CCGGTAGAAG ATTTATTTTGCTACACTTAT ATCTACAACA 3000 AAATCAACTT ATGAAATTGT CTTGGAAACT ACCTCTAACCCGGTAGAATG AATTTGAATG 3060 AAAATTAAAC CAACTTACGG AATCGCCCAA CATATGTCGATTAAAGTGGA TATGGATACA 3120 TATGAAGAAG CCCTAGAGAT AATCTAAATG GTTTCAGAATTGAGGGTTAT TTTTTGAAGT 3180 TTGATGGGAA GATAAGACCA TAACGGTAGT TCACAGAGATAAAAGGGTTA TTTTTTTCAG 3240 AAATATTTGT GCTGCAATTG ATCCTGTGCC TCAAATTCAGCCTGCAACCA AGGCCAGGTT 3300 CTAGAGCGAA CAAGGCCCAC GTCACCCGTG GCCCGTCAGGCGAAGCAGGT CTTGTGCAGA 3360 CTTTGAGAGG GATTGGATAT CAACGGAACC AATCACGCACGGCAATGCGA TTCCCAGCCC 3420 ACCTGTAACG TTCCAGTGGG CCATCCTTAA CTCCAAGCCCAACGGCCCTA CCCCATCTCG 3480 TCGTGTCATC CACTCCGCCG CACAGGCGCT CAGCTCCGCAACGCCGCCGG AAATGGTCGC 3540 CGCCACAGCC ACCGCCATGG CCACCGCTGC ATCGCCGCTACTCAACGGGA CCCGAATACC 3600 TGCGCGGCTC CGCCATCGAG GACTCAGCGT GCGCTGCGCTGCTGTGGCGG GCGGCGCGGC 3660 CGAGGCACCG GCATCCACCG GCGCGCGGCT GTCCGCGGACTGCGTTGTGG TGGGCGGAGG 3720 CATCAGTGGC CTCTGCACCG CGCAGGCGCT GGCCACGCGGCACGGCGTCG GGGACGTGCT 3780 TGTCACGGAG GCCCGCGCCC GCCCCGGCGG CAACATTACCACCGTCGAGC GCCCCGAGGA 3840 AGGGTACC 3848 1826 base pairs nucleic acidsingle linear cDNA NO NO Gossypium hirsutum (cotton) pWDC-15 (NRRLB-21594) misc_feature 31..1647 /product= “Cotton protox-1 codingsequence” 15 CCTCTCGCTC GCCTGGCCCC ACCACCAATC ATGACGGCTC TAATCGACCTTTCTCTTCTC 60 CGTTCCTCGC CCTCCGTTTC CCCTTTCTCC ATACCCCACC ACCAGCATCCGCCCCGCTTT 120 CGTAAACCTT TCAAGCTCCG ATGCTCCCTC GCCGAGGGTC CCACGATTTCCTCATCTAAA 180 ATCGACGGGG GAGAATCATC CATCGCGGAT TGCGTCATCG TTGGAGGTGGTATCAGTGGA 240 CTTTGCATTG CTCAAGCTCT CGCCACCAAG CACCGTGACG TCGCTTCCAATGTGATTGTG 300 ACGGAGGCCA GAGACCGTGT TGGTGGCAAC ATCACTACCG TTGAGAGAGATGGATATCTG 360 TGGGAAGAAG GCCCCAACAG TTTTCAGCCC TCCGATCCTA TTCTAACCATGGCCGTGGAT 420 AGTGGATTGA AGGACGATTT GGTTTTAGGT GACCCTAATG CACCGCGATTTGTACTATGG 480 GAGGGAAAAC TAAGGCCTGT GCCCTCCAAG CCAACCGACT TGCCGTTTTTTGATTTGATG 540 AGCATTGCTG GAAAACTTAG GGCTGGGTTC GGGGCTATTG GCATTCGGCCTCCCCCTCCG 600 GGTTATGAAG AATCGGTGGA GGAGTTTGTG CGCCGTAATC TTGGTGCTGAGGTTTTTGAA 660 CGCTTTATTG AACCATTTTG TTCAGGTGTT TATGCAGGGG ATCCTTCAAAATTAAGCATG 720 AAAGCAGCAT TTGGAAGAGT ATGGAAGCTA GAAGAGATTG GTGGCAGCATCATTGGTGGC 780 ACTTTCAAGA CAATCCAGGA GAGAAATAAG ACACCTAAGC CACCCAGAGACCCGCGTCTG 840 CCAAAACCGA AGGGCCAAAC AGTTGGATCT TTTAGGAAGG GACTTACCATGCTGCCTGAG 900 GCAATTGCTA ACAGTTTGGG TAGCAATGTA AAATTATCTT GGAAGCTTTCCAGTATTACC 960 AAATTGGGCA ATGGAGGGTA TAACTTGACA TTTGAAACAC CTGAAGGAATGGTATCTCTT 1020 CAGAGTAGAA GTGTTGTAAT GACCATTCCA TCCCATGTTG CCAGTAACTTGTTGCATCCT 1080 CTCTCGGCTG CTGCTGCAGA TGCATTATCC CAATTTTATT ATCCTCCAGTTGCATCAGTC 1140 ACAGTCTCCT ATCCAAAAGA AGCCATTCGA AAAGAATGTT TGATTGATGGTGAACTTAAG 1200 GGGTTTGGCC AGTTGCACCC ACGCAGCCAA GGAATTGAAA CTTTAGGGACGATATACAGT 1260 TCATCACTTT TCCCCAATCG AGCTCCATCT GGCAGGGTGT TGCTCTTGAACTACATAGGA 1320 GGAGCTACCA ACACTGGAAT TTTGTCCAAG ACTGAAGGGG AACTTGTAGAAGCAGTTGAT 1380 CGTGATTTGA GAAAAATGCT TATAAATCCT AATGCAAAGG ATCCTCTTGTTTTGGGTGTA 1440 AGAGTATGGC CAAAAGCCAT TCCACAGTTC TTGGTTGGTC ATTTGGATCTCCTTGATAGT 1500 GCAAAAATGG CTCTCAGGGA TTCTGGGTTT CATGGACTGT TTCTTGGGGGCAACTATGTA 1560 TCTGGTGTGG CATTAGGACG GTGTGTGGAA GGTGCTTACG AGGTTGCAGCTGAAGTGAAG 1620 GAATTCCTGT CACAATATGC ATACAAATAA TATTGAAATT CTTGTCAGGCTGCAAATGTA 1680 GAAGTCAGTT ATTGGATAGT ATCTCTTTAG CTAAAAAATT GGGTAGGGTTTTTTTTGTTA 1740 GTTCCTTGAC CACTTTTTGG GGTTTTCATT AGAACTTCAT ATTTGTATATCATGTTGCAA 1800 TATCAAAAAA AAAAAAAAAA AAAAAA 1826 539 amino acids aminoacid Not Relevant Not Relevant protein not provided 16 Met Thr Ala LeuIle Asp Leu Ser Leu Leu Arg Ser Ser Pro Ser Val 1 5 10 15 Ser Pro PheSer Ile Pro His His Gln His Pro Pro Arg Phe Arg Lys 20 25 30 Pro Phe LysLeu Arg Cys Ser Leu Ala Glu Gly Pro Thr Ile Ser Ser 35 40 45 Ser Lys IleAsp Gly Gly Glu Ser Ser Ile Ala Asp Cys Val Ile Val 50 55 60 Gly Gly GlyIle Ser Gly Leu Cys Ile Ala Gln Ala Leu Ala Thr Lys 65 70 75 80 His ArgAsp Val Ala Ser Asn Val Ile Val Thr Glu Ala Arg Asp Arg 85 90 95 Val GlyGly Asn Ile Thr Thr Val Glu Arg Asp Gly Tyr Leu Trp Glu 100 105 110 GluGly Pro Asn Ser Phe Gln Pro Ser Asp Pro Ile Leu Thr Met Ala 115 120 125Val Asp Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Asn Ala 130 135140 Pro Arg Phe Val Leu Trp Glu Gly Lys Leu Arg Pro Val Pro Ser Lys 145150 155 160 Pro Thr Asp Leu Pro Phe Phe Asp Leu Met Ser Ile Ala Gly LysLeu 165 170 175 Arg Ala Gly Phe Gly Ala Ile Gly Ile Arg Pro Pro Pro ProGly Tyr 180 185 190 Glu Glu Ser Val Glu Glu Phe Val Arg Arg Asn Leu GlyAla Glu Val 195 200 205 Phe Glu Arg Phe Ile Glu Pro Phe Cys Ser Gly ValTyr Ala Gly Asp 210 215 220 Pro Ser Lys Leu Ser Met Lys Ala Ala Phe GlyArg Val Trp Lys Leu 225 230 235 240 Glu Glu Ile Gly Gly Ser Ile Ile GlyGly Thr Phe Lys Thr Ile Gln 245 250 255 Glu Arg Asn Lys Thr Pro Lys ProPro Arg Asp Pro Arg Leu Pro Lys 260 265 270 Pro Lys Gly Gln Thr Val GlySer Phe Arg Lys Gly Leu Thr Met Leu 275 280 285 Pro Glu Ala Ile Ala AsnSer Leu Gly Ser Asn Val Lys Leu Ser Trp 290 295 300 Lys Leu Ser Ser IleThr Lys Leu Gly Asn Gly Gly Tyr Asn Leu Thr 305 310 315 320 Phe Glu ThrPro Glu Gly Met Val Ser Leu Gln Ser Arg Ser Val Val 325 330 335 Met ThrIle Pro Ser His Val Ala Ser Asn Leu Leu His Pro Leu Ser 340 345 350 AlaAla Ala Ala Asp Ala Leu Ser Gln Phe Tyr Tyr Pro Pro Val Ala 355 360 365Ser Val Thr Val Ser Tyr Pro Lys Glu Ala Ile Arg Lys Glu Cys Leu 370 375380 Ile Asp Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Ser Gln 385390 395 400 Gly Ile Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe ProAsn 405 410 415 Arg Ala Pro Ser Gly Arg Val Leu Leu Leu Asn Tyr Ile GlyGly Ala 420 425 430 Thr Asn Thr Gly Ile Leu Ser Lys Thr Glu Gly Glu LeuVal Glu Ala 435 440 445 Val Asp Arg Asp Leu Arg Lys Met Leu Ile Asn ProAsn Ala Lys Asp 450 455 460 Pro Leu Val Leu Gly Val Arg Val Trp Pro LysAla Ile Pro Gln Phe 465 470 475 480 Leu Val Gly His Leu Asp Leu Leu AspSer Ala Lys Met Ala Leu Arg 485 490 495 Asp Ser Gly Phe His Gly Leu PheLeu Gly Gly Asn Tyr Val Ser Gly 500 505 510 Val Ala Leu Gly Arg Cys ValGlu Gly Ala Tyr Glu Val Ala Ala Glu 515 520 525 Val Lys Glu Phe Leu SerGln Tyr Ala Tyr Lys 530 535 1910 base pairs nucleic acid single linearcDNA NO NO Beta vulgaris (Sugar Beet) pWDC-16 (NRRL B-21595N)misc_feature 1..1680 /product= “Sugar Beet protox-1 coding sequence” 17ATGAAATCAA TGGCGTTATC AAACTGCATT CCACAGACAC AGTGCATGCC ATTGCGCAGC 60AGCGGGCATT ACAGGGGTAA TTGTATCATG TTGTCAATTC CATGTAGTTT AATTGGAAGA 120CGAGGTTATT ATTCACATAA GAAGAGGAGG ATGAGCATGA GTTGCAGCAC AAGCTCAGGC 180TCAAAGTCAG CGGTTAAAGA AGCAGGATCA GGATCAGGTG CAGGAGGATT GCTAGACTGC 240GTAATCGTTG GAGGTGGAAT TAGCGGGCTT TGCATCGCGC AGGCTCTTTG TACAAAACAC 300TCCTCTTCCT CTTTATCCCC AAATTTTATA GTTACAGAGG CCAAAGACAG AGTTGGCGGC 360AACATCGTCA CTGTGGAGGC CGATGGCTAT ATCTGGGAGG AGGGACCCAA TAGCTTCCAG 420CCTTCCGACG CGGTGCTCAC CATGGCGGTC GACAGTGGCT TGAAAGATGA GTTGGTGCTC 480GGAGATCCCA ATGCTCCTCG CTTTGTGCTA TGGAATGACA AATTAAGGCC CGTACCTTCC 540AGTCTCACCG ACCTCCCTTT CTTCGACCTC ATGACCATTC CGGGCAAGAT TAGGGCTGCT 600CTTGGTGCTC TCGGATTTCG CCCTTCTCCT CCACCTCATG AGGAATCTGT TGAACACTTT 660GTGCGTCGTA ATCTCGGAGA TGAGGTCTTT GAACGCTTGA TTGAACCCTT TTGTTCAGGT 720GTGTATGCCG GTGATCCTGC CAAGCTGAGT ATGAAAGCTG CTTTTGGGAA GGTCTGGAAG 780TTGGAGCAAA AGGGTGGCAG CATAATTGGT GGCACTCTCA AAGCTATACA GGAAAGAGGG 840AGTAATCCTA AGCCGCCCCG TGACCAGCGC CTCCCTAAAC CAAAGGGTCA GACTGTTGGA 900TCCTTTAGAA AGGGACTCGT TATGTTGCCT ACCGCCATTT CTGCTCGACT TGGCAGTAGA 960GTGAAACTAT CTTGGACCCT TTCTAGTATC GTAAAGTCAC TCAATGGAGA ATATAGTCTG 1020ACTTATGATA CCCCAGATGG CTTGGTTTCT GTAAGAACCA AAAGTGTTGT GATGACTGTT 1080CCATCATATG TTGCAAGTAG GCTTCTTCGT CCACTTTCAG ACTCTGCTGC AGATTCTCTT 1140TCAAAATTTT ACTATCCACC AGTTGCAGCA GTGTCACTTT CCTATCCTAA AGAAGCGATC 1200AGATCAGAAT GCTTGATTAA TGGTGAACTT CAAGGTTTCG GGCAACTACA TCCCCGCAGT 1260CAGGGTGTGG AAACCTTGGG AACAATTTAT AGTTCGTCTC TTTTCCCTGG TCGAGCACCA 1320CCTGGTAGGA TCTTGATCTT GAGCTACATC GGAGGTGCTA AAAATCCTGG CATATTAAAC 1380AAGTCGAAAG ATGAACTTGC CAAGACAGTT GACAAGGACC TGAGAAGAAT GCTTATAAAT 1440CCTGATGCAA AACTTCCTCG TGTACTGGGT GTGAGAGTAT GGCCTCAAGC AATACCCCAG 1500TTTTCTATTG GGCACTTTGA TCTGCTCGAT GCTGCAAAAG CTGCTCTGAC AGATACAGGG 1560GTCAAAGGAC TGTTTCTTGG TGGCAACTAT GTTTCAGGTG TTGCCTTGGG GCGGTGTATA 1620GAGGGTGCTT ATGAGTCTGC AGCTGAGGTA GTAGATTTCC TCTCACAGTA CTCAGACAAA 1680TAGAGCTTCA GCATCCTGTG TAATTCAACA CAGGCCTTTT TGTATCTGTT GTGCGCGCAT 1740GTAGTCTGGT CGTGGTGCTA GGATTGATTA GTTGCTCTGC TGTGTGATCC ACAAGAATTT 1800TGATGGAATT TTTCCAGATG TGGGCATTAT ATGTTGCTGT CTTATAAATC CTTAATTTGT 1860ACGTTTAGTG AATTACACCG CATTTGATGA CTAAAAAAAA AAAAAAAAAA 1910 560 aminoacids amino acid Not Relevant Not Relevant protein not provided 18 MetLys Ser Met Ala Leu Ser Asn Cys Ile Pro Gln Thr Gln Cys Met 1 5 10 15Pro Leu Arg Ser Ser Gly His Tyr Arg Gly Asn Cys Ile Met Leu Ser 20 25 30Ile Pro Cys Ser Leu Ile Gly Arg Arg Gly Tyr Tyr Ser His Lys Lys 35 40 45Arg Arg Met Ser Met Ser Cys Ser Thr Ser Ser Gly Ser Lys Ser Ala 50 55 60Val Lys Glu Ala Gly Ser Gly Ser Gly Ala Gly Gly Leu Leu Asp Cys 65 70 7580 Val Ile Val Gly Gly Gly Ile Ser Gly Leu Cys Ile Ala Gln Ala Leu 85 9095 Cys Thr Lys His Ser Ser Ser Ser Leu Ser Pro Asn Phe Ile Val Thr 100105 110 Glu Ala Lys Asp Arg Val Gly Gly Asn Ile Val Thr Val Glu Ala Asp115 120 125 Gly Tyr Ile Trp Glu Glu Gly Pro Asn Ser Phe Gln Pro Ser AspAla 130 135 140 Val Leu Thr Met Ala Val Asp Ser Gly Leu Lys Asp Glu LeuVal Leu 145 150 155 160 Gly Asp Pro Asn Ala Pro Arg Phe Val Leu Trp AsnAsp Lys Leu Arg 165 170 175 Pro Val Pro Ser Ser Leu Thr Asp Leu Pro PhePhe Asp Leu Met Thr 180 185 190 Ile Pro Gly Lys Ile Arg Ala Ala Leu GlyAla Leu Gly Phe Arg Pro 195 200 205 Ser Pro Pro Pro His Glu Glu Ser ValGlu His Phe Val Arg Arg Asn 210 215 220 Leu Gly Asp Glu Val Phe Glu ArgLeu Ile Glu Pro Phe Cys Ser Gly 225 230 235 240 Val Tyr Ala Gly Asp ProAla Lys Leu Ser Met Lys Ala Ala Phe Gly 245 250 255 Lys Val Trp Lys LeuGlu Gln Lys Gly Gly Ser Ile Ile Gly Gly Thr 260 265 270 Leu Lys Ala IleGln Glu Arg Gly Ser Asn Pro Lys Pro Pro Arg Asp 275 280 285 Gln Arg LeuPro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe Arg Lys 290 295 300 Gly LeuVal Met Leu Pro Thr Ala Ile Ser Ala Arg Leu Gly Ser Arg 305 310 315 320Val Lys Leu Ser Trp Thr Leu Ser Ser Ile Val Lys Ser Leu Asn Gly 325 330335 Glu Tyr Ser Leu Thr Tyr Asp Thr Pro Asp Gly Leu Val Ser Val Arg 340345 350 Thr Lys Ser Val Val Met Thr Val Pro Ser Tyr Val Ala Ser Arg Leu355 360 365 Leu Arg Pro Leu Ser Asp Ser Ala Ala Asp Ser Leu Ser Lys PheTyr 370 375 380 Tyr Pro Pro Val Ala Ala Val Ser Leu Ser Tyr Pro Lys GluAla Ile 385 390 395 400 Arg Ser Glu Cys Leu Ile Asn Gly Glu Leu Gln GlyPhe Gly Gln Leu 405 410 415 His Pro Arg Ser Gln Gly Val Glu Thr Leu GlyThr Ile Tyr Ser Ser 420 425 430 Ser Leu Phe Pro Gly Arg Ala Pro Pro GlyArg Ile Leu Ile Leu Ser 435 440 445 Tyr Ile Gly Gly Ala Lys Asn Pro GlyIle Leu Asn Lys Ser Lys Asp 450 455 460 Glu Leu Ala Lys Thr Val Asp LysAsp Leu Arg Arg Met Leu Ile Asn 465 470 475 480 Pro Asp Ala Lys Leu ProArg Val Leu Gly Val Arg Val Trp Pro Gln 485 490 495 Ala Ile Pro Gln PheSer Ile Gly His Phe Asp Leu Leu Asp Ala Ala 500 505 510 Lys Ala Ala LeuThr Asp Thr Gly Val Lys Gly Leu Phe Leu Gly Gly 515 520 525 Asn Tyr ValSer Gly Val Ala Leu Gly Arg Cys Ile Glu Gly Ala Tyr 530 535 540 Glu SerAla Ala Glu Val Val Asp Phe Leu Ser Gln Tyr Ser Asp Lys 545 550 555 5601784 base pairs nucleic acid single linear cDNA NO NO Brassica napus(oilseed rape) pWDC-17 (NRRL B-21615) misc_feature 47..1654 /product=“Oilseed rape protox-1 coding sequence” 19 GGGCCCCCCC CAAAATTGAGGATTCTCCTT CTCGCGGGCG ATCGCCATGG ATTTATCTCT 60 TCTCCGTCCG CAGCCATTCCTATCGCCATT CTCAAATCCA TTTCCTCGGT CGCGTCCCTA 120 CAAGCCTCTC AACCTCCGTTGCTCCGTATC CGGTGGATCC GTCGTCGGCT CTTCTACAAT 180 CGAAGGCGGA GGAGGAGGTAAAACCGTCAC GGCGGACTGC GTGATCGTCG GCGGAGGAAT 240 CAGCGGCCTG TGCATTGCGCAAGCGCTCGT GACGAAGCAC CCAGACGCTG CAAAGAATGT 300 GATGGTGACG GAGGCGAAGGACCGTGTGGG AGGGAATATC ATCACGCGAG AGGAGCAAGG 360 GTTTCTATGG GAAGAAGGTCCCAATAGCTT TCAGCCGTCT GATCCTATGC TCACTATGGT 420 GGTAGATAGT GGTTTGAAAGATGATCTAGT CTTGGGAGAT CCTACTGCTC CGAGGTTTGT 480 GTTGTGGAAT GGGAAGCTGAGGCCGGTTCC GTCGAAGCTA ACTGACTTGC CTTTCTTTGA 540 CTTGATGAGT ATTGGAGGGAAGATTAGAGC TGGGTTTGGT GCCATTGGTA TTCGACCTTC 600 ACCTCCGGGT CGTGAGGAATCAGTGGAAGA GTTTGTAAGG CGTAATCTTG GTGATGAGGT 660 TTTTGAGCGC TTGATTGAACCCTTTTGCTC AGGTGTTTAT GCGGGAGATC CTGCGAAACT 720 GAGTATGAAA GCAGCTTTTGGGAAGGTTTG GAAGCTAGAG GAGAATGGTG GGAGCATCAT 780 TGGTGGTGCT TTTAAGGCAATTCAAGCGAA AAATAAAGCT CCCAAGACAA CCCGAGATCC 840 GCGTCTGCCA AAGCCAAAGGGCCAAACTGT TGGTTCTTTC AGGAAAGGAC TCACAATGCT 900 GCCAGAGGCA ATCTCCGCAAGGTTGGGTGA CAAGGTGAAA GTTTCTTGGA AGCTCTCAAG 960 TATCACTAAG CTGGCCAGCGGAGAATATAG CTTAACTTAC GAAACTCCGG AGGGTATAGT 1020 CACTGTACAG AGCAAAAGTGTAGTGATGAC TGTGCCATCT CATGTTGCTA GTAGTCTCTT 1080 GCGCCCTCTC TCTGATTCTGCAGCTGAAGC GCTCTCAAAA CTCTACTATC CGCCAGTTGC 1140 AGCCGTATCC ATCTCATACGCGAAAGAAGC AATCCGAAGC GAATGCTTAA TAGATGGTGA 1200 ACTAAAAGGG TTCGGCCAGTTGCATCCACG CACGCAAAAA GTGGAAACTC TTGGAACAAT 1260 ATACAGTTCA TCGCTCTTTCCCAACCGAGC ACCGCCTGGA AGAGTATTGC TATTGAACTA 1320 CATCGGTGGA GCTACCAACACTGGGATCTT ATCAAAGTCG GAAGGTGAGT TAGTGGAAGC 1380 AGTAGATAGA GACTTGAGGAAGATGCTGAT AAAGCCAAGC TCGACCGATC CACTTGTACT 1440 TGGAGTAAAA TTATGGCCTCAAGCCATTCC TCAGTTTCTG ATAGGTCACA TTGATTTGGT 1500 AGACGCAGCG AAAGCATCGCTCTCGTCATC TGGTCATGAG GGCTTATTCT TGGGTGGAAA 1560 TTACGTTGCC GGTGTAGCATTGGGTCGGTG TGTGGAAGGT GCTTATGAAA CTGCAACCCA 1620 AGTGAATGAT TTCATGTCAAGGTATGCTTA CAAGTAATGT AACGCAGCAA CGATTTGATA 1680 CTAAGTAGTA GATTTTGCAGTTTTGACTTT AAGAACACTC TGTTTGTGAA AAATTCAAGT 1740 CTGTGATTGA GTAAATTTATGTATTATTAC TAAAAAAAAA AAAA 1784 536 amino acids amino acid Not RelevantNot Relevant protein not provided 20 Met Asp Leu Ser Leu Leu Arg Pro GlnPro Phe Leu Ser Pro Phe Ser 1 5 10 15 Asn Pro Phe Pro Arg Ser Arg ProTyr Lys Pro Leu Asn Leu Arg Cys 20 25 30 Ser Val Ser Gly Gly Ser Val ValGly Ser Ser Thr Ile Glu Gly Gly 35 40 45 Gly Gly Gly Lys Thr Val Thr AlaAsp Cys Val Ile Val Gly Gly Gly 50 55 60 Ile Ser Gly Leu Cys Ile Ala GlnAla Leu Val Thr Lys His Pro Asp 65 70 75 80 Ala Ala Lys Asn Val Met ValThr Glu Ala Lys Asp Arg Val Gly Gly 85 90 95 Asn Ile Ile Thr Arg Glu GluGln Gly Phe Leu Trp Glu Glu Gly Pro 100 105 110 Asn Ser Phe Gln Pro SerAsp Pro Met Leu Thr Met Val Val Asp Ser 115 120 125 Gly Leu Lys Asp AspLeu Val Leu Gly Asp Pro Thr Ala Pro Arg Phe 130 135 140 Val Leu Trp AsnGly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr Asp 145 150 155 160 Leu ProPhe Phe Asp Leu Met Ser Ile Gly Gly Lys Ile Arg Ala Gly 165 170 175 PheGly Ala Ile Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu Ser 180 185 190Val Glu Glu Phe Val Arg Arg Asn Leu Gly Asp Glu Val Phe Glu Arg 195 200205 Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ala Lys 210215 220 Leu Ser Met Lys Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Glu Asn225 230 235 240 Gly Gly Ser Ile Ile Gly Gly Ala Phe Lys Ala Ile Gln AlaLys Asn 245 250 255 Lys Ala Pro Lys Thr Thr Arg Asp Pro Arg Leu Pro LysPro Lys Gly 260 265 270 Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Thr MetLeu Pro Glu Ala 275 280 285 Ile Ser Ala Arg Leu Gly Asp Lys Val Lys ValSer Trp Lys Leu Ser 290 295 300 Ser Ile Thr Lys Leu Ala Ser Gly Glu TyrSer Leu Thr Tyr Glu Thr 305 310 315 320 Pro Glu Gly Ile Val Thr Val GlnSer Lys Ser Val Val Met Thr Val 325 330 335 Pro Ser His Val Ala Ser SerLeu Leu Arg Pro Leu Ser Asp Ser Ala 340 345 350 Ala Glu Ala Leu Ser LysLeu Tyr Tyr Pro Pro Val Ala Ala Val Ser 355 360 365 Ile Ser Tyr Ala LysGlu Ala Ile Arg Ser Glu Cys Leu Ile Asp Gly 370 375 380 Glu Leu Lys GlyPhe Gly Gln Leu His Pro Arg Thr Gln Lys Val Glu 385 390 395 400 Thr LeuGly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro 405 410 415 ProGly Arg Val Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn Thr 420 425 430Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala Val Asp Arg 435 440445 Asp Leu Arg Lys Met Leu Ile Lys Pro Ser Ser Thr Asp Pro Leu Val 450455 460 Leu Gly Val Lys Leu Trp Pro Gln Ala Ile Pro Gln Phe Leu Ile Gly465 470 475 480 His Ile Asp Leu Val Asp Ala Ala Lys Ala Ser Leu Ser SerSer Gly 485 490 495 His Glu Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala GlyVal Ala Leu 500 505 510 Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala ThrGln Val Asn Asp 515 520 525 Phe Met Ser Arg Tyr Ala Tyr Lys 530 535 1224base pairs nucleic acid single linear cDNA NO NO Oryza sative (rice)pWDC-18 (NRRL B-21648) misc_feature 1..936 /product= “Rice protox-1partial coding sequence” 21 CGGGCTTTGA AGGCTGCATT TGGGAAGGTG TGGAGGCTGGAGGATACTGG AGGTAGCATT 60 ATTGGTGGAA CCATCAAGAC AATCCAGGAG AGGGGGAAAAACCCCAAACC GCCGAGGGAT 120 CCCCGCCTTC CAACGCCAAA GGGGCAGACA GTTGCATCTTTCAGGAAGGG TCTGACTATG 180 CTCCCGGATG CTATTACATC TAGGTTGGGT AGCAAAGTCAAACTTTCATG GAAGTTGACA 240 AGCATTACAA AGTCAGACAA CAAAGGATAT GCATTAGTGTATGAAACACC AGAAGGGGTG 300 GTCTCGGTGC AAGCTAAAAC TGTTGTCATG ACCATCCCATCATATGTTGC TAGTGATATC 360 TTGCGGCCAC TTTCAAGTGA TGCAGCAGAT GCTCTGTCAATATTCTATTA TCCACCAGTT 420 GCTGCTGTAA CTGTTTCATA TCCAAAAGAA GCAATTAGAAAAGAATGCTT AATTGACGGA 480 GAGCTCCAGG GTTTCGGCCA GCTGCATCCG CGTAGTCAGGGAGTTGAGAC TTTAGGAACA 540 ATATATAGCT CATCACTCTT TCCAAATCGT GCTCCAGCTGGAAGGGTGTT ACTTCTGAAC 600 TACATAGGAG GTTCTACAAA TACAGGGATT GTTTCCAAGACTGAAAGTGA GCTGGTAGAC 660 GCAGTTGACC GTGACCTCAG GAAGATGCTG ATAAATCCTAGAGCAGTGGA CCCTTTGGTC 720 CTTGGCGTCC GGGTATGGCC ACAAGCCATA CCACAGTTCCTCATTGGCCA TCTTGATCAT 780 CTTGAGGCTG CAAAATCTGC CCTGGGCAAA GGTGGGTATGATGGATTGTT CCTCGGAGGG 840 AACTATGTTG CAGGAGTTGC CCTGGGCCGA TGCGTTGAAGGTGCATATGA GAGTGCCTCA 900 CAAATATCTG ACTACTTGAC CAAGTACGCC TACAAGTGATCAAAGTTGGC CTGCTCCTTT 960 TGGCACATAG ATGTGAGGCT TCTAGCAGCA AAAATTTCATGGGCATCTTT TTATCCTGAT 1020 TCTAATTAGT TAGAATTTAG AATTGTAGAG GAATGTTCCATTTGCAGTTC ATAATAGTTG 1080 TTCAGATTTC AGCCATTCAA TTTGTGCAGC CATTTACTATATGTAGTATG ATCTTGTAAG 1140 TACTACTAAG AACAAATCAA TTATATTTTC CTGCAAGTGACATCTTAATC GTCAGCAAAT 1200 CCAGTTACTA GTAAAAAAAA AAAA 1224 312 aminoacids amino acid Not Relevant Not Relevant protein not provided 22 ArgAla Leu Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Asp Thr 1 5 10 15Gly Gly Ser Ile Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Gly 20 25 30Lys Asn Pro Lys Pro Pro Arg Asp Pro Arg Leu Pro Thr Pro Lys Gly 35 40 45Gln Thr Val Ala Ser Phe Arg Lys Gly Leu Thr Met Leu Pro Asp Ala 50 55 60Ile Thr Ser Arg Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu Thr 65 70 7580 Ser Ile Thr Lys Ser Asp Asn Lys Gly Tyr Ala Leu Val Tyr Glu Thr 85 9095 Pro Glu Gly Val Val Ser Val Gln Ala Lys Thr Val Val Met Thr Ile 100105 110 Pro Ser Tyr Val Ala Ser Asp Ile Leu Arg Pro Leu Ser Ser Asp Ala115 120 125 Ala Asp Ala Leu Ser Ile Phe Tyr Tyr Pro Pro Val Ala Ala ValThr 130 135 140 Val Ser Tyr Pro Lys Glu Ala Ile Arg Lys Glu Cys Leu IleAsp Gly 145 150 155 160 Glu Leu Gln Gly Phe Gly Gln Leu His Pro Arg SerGln Gly Val Glu 165 170 175 Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu PhePro Asn Arg Ala Pro 180 185 190 Ala Gly Arg Val Leu Leu Leu Asn Tyr IleGly Gly Ser Thr Asn Thr 195 200 205 Gly Ile Val Ser Lys Thr Glu Ser GluLeu Val Glu Ala Val Asp Arg 210 215 220 Asp Leu Arg Lys Met Leu Ile AsnPro Arg Ala Val Asp Pro Leu Val 225 230 235 240 Leu Gly Val Arg Val TrpPro Gln Ala Ile Pro Gln Phe Leu Ile Gly 245 250 255 His Leu Asp His LeuGlu Ala Ala Lys Ser Ala Leu Gly Lys Gly Gly 260 265 270 Tyr Asp Gly LeuPhe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala Leu 275 280 285 Gly Arg CysVal Glu Gly Ala Tyr Glu Ser Ala Ser Gln Ile Ser Asp 290 295 300 Tyr LeuThr Lys Tyr Ala Tyr Lys 305 310 1590 base pairs nucleic acid singlelinear cDNA NO NO Sorghum bicolor (sorghum) pWDC-19 (NRRL B-21649)misc_feature 1..1320 /product= “Sorghum protox-1 partial codingsequence” 23 TCCACCGTCG AGCGCCCCGA GGAAGGGTAC CTCTGGGAGG AGGGTCCCAACAGCTTCCAG 60 CCATCCGACC CCGTTCTCTC CATGGCCGTG GACAGCGGGC TGAAGGATGACCTGGTTTTT 120 GGGGACCCCA ACGCGCCACG GTTCGTGCTG TGGGAGGGGA AGCTGAGGCCCGTGCCATCC 180 AAGCCCGCCG ACCTCCCGTT CTTCGATCTC ATGAGCATCC CTGGCAAGCTCAGGGCCGGT 240 CTCGGCGCGC TTGGCATCCG CCCGCCTGCT CCAGGCCGCG AGGAGTCAGTGGAGGAGTTT 300 GTGCGCCGCA ACCTCGGTGC TGAGGTCTTT GAGCGCCTAA TTGAGCCTTTCTGCTCAGGT 360 GTCTATGCTG GCGATCCTTC CAAGCTCAGT ATGAAGGCTG CATTTGGGAAGGTGTGGCGG 420 TTAGAAGAAG CTGGAGGTAG TATTATTGGT GGAACCATCA AGACGATTCAGGAGAGGGGC 480 AAGAATCCAA AACCACCGAG GGATCCCCGC CTTCCGAAGC CAAAAGGGCAGACAGTTGCA 540 TCTTTCAGGA AGGGTCTTGC CATGCTTCCA AATGCCATCA CATCCAGCTTGGGTAGTAAA 600 GTCAAACTAT CATGGAAACT CACGAGCATG ACAAAATCAG ATGGCAAGGGGTATGTTTTA 660 GAGTATGAAA CACCAGAAGG GGTTGTTTTG GTGCAGGCTA AAAGTGTTATCATGACCATT 720 CCATCATATG TTGCTAGCGA CATTTTGCGT CCACTTTCAG GTGATGCTGCAGATGTTCTA 780 TCAAGATTCT ATTATCCACC AGTTGCTGCT GTAACGGTTT CGTATCCAAAGGAAGCAATT 840 AGAAAAGAAT GCTTAATTGA TGGGGAACTC CAGGGTTTTG GCCAGTTGCATCCACGTAGT 900 CAAGGAGTTG AGACATTAGG AACAATATAC AGCTCATCAC TCTTTCCAAATCGTGCTCCT 960 GCTGGTAGGG TGTTACTTCT AAACTACATA GGAGGTGCTA CAAACACAGGAATTGTTTCC 1020 AAGACTGAAA GTGAGCTGGT AGAAGCAGTT GACCGTGACC TCCGAAAAATGCTTATAAAT 1080 CCTACAGCAG TGGACCCTTT AGTCCTTGGT GTCCGAGTTT GGCCACAAGCCATACCTCAG 1140 TTCCTGGTAG GACATCTTGA TCTTCTGGAG GCCGCAAAAT CTGCCCTGGACCAAGGTGGC 1200 TATAATGGGC TGTTCCTAGG AGGGAACTAT GTTGCAGGAG TTGCCCTGGGCAGATGCATT 1260 GAGGGCGCAT ATGAGAGTGC CGCGCAAATA TATGACTTCT TGACCAAGTACGCCTACAAG 1320 TGATGGAAGA AGTGGAGCGC TGCTTGTTAA TTGTTATGTT GCATAGATGAGGTGAGACCA 1380 GGAGTAGTAA AAGGCGTCAC GAGTATTTTT CATTCTTATT TTGTAAATTGCACTTCTGTT 1440 TTTTTTTCCT GTCAGTAATT AGTTAGATTT TAGTTATGTA GGAGATTGTTGTGTTCACTG 1500 CCCTACAAAA GAATTTTTAT TTTGCATTCG TTTATGAGAG CTGTGCAGACTTATGTAACG 1560 TTTTACTGTA AGTATCAACA AAATCAAATA 1590 440 amino acidsamino acid Not Relevant Not Relevant protein not provided 24 Ser Thr ValGlu Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro 1 5 10 15 Asn SerPhe Gln Pro Ser Asp Pro Val Leu Ser Met Ala Val Asp Ser 20 25 30 Gly LeuLys Asp Asp Leu Val Phe Gly Asp Pro Asn Ala Pro Arg Phe 35 40 45 Val LeuTrp Glu Gly Lys Leu Arg Pro Val Pro Ser Lys Pro Ala Asp 50 55 60 Leu ProPhe Phe Asp Leu Met Ser Ile Pro Gly Lys Leu Arg Ala Gly 65 70 75 80 LeuGly Ala Leu Gly Ile Arg Pro Pro Ala Pro Gly Arg Glu Glu Ser 85 90 95 ValGlu Glu Phe Val Arg Arg Asn Leu Gly Ala Glu Val Phe Glu Arg 100 105 110Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser Lys 115 120125 Leu Ser Met Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Glu Ala 130135 140 Gly Gly Ser Ile Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Gly145 150 155 160 Lys Asn Pro Lys Pro Pro Arg Asp Pro Arg Leu Pro Lys ProLys Gly 165 170 175 Gln Thr Val Ala Ser Phe Arg Lys Gly Leu Ala Met LeuPro Asn Ala 180 185 190 Ile Thr Ser Ser Leu Gly Ser Lys Val Lys Leu SerTrp Lys Leu Thr 195 200 205 Ser Met Thr Lys Ser Asp Gly Lys Gly Tyr ValLeu Glu Tyr Glu Thr 210 215 220 Pro Glu Gly Val Val Leu Val Gln Ala LysSer Val Ile Met Thr Ile 225 230 235 240 Pro Ser Tyr Val Ala Ser Asp IleLeu Arg Pro Leu Ser Gly Asp Ala 245 250 255 Ala Asp Val Leu Ser Arg PheTyr Tyr Pro Pro Val Ala Ala Val Thr 260 265 270 Val Ser Tyr Pro Lys GluAla Ile Arg Lys Glu Cys Leu Ile Asp Gly 275 280 285 Glu Leu Gln Gly PheGly Gln Leu His Pro Arg Ser Gln Gly Val Glu 290 295 300 Thr Leu Gly ThrIle Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro 305 310 315 320 Ala GlyArg Val Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn Thr 325 330 335 GlyIle Val Ser Lys Thr Glu Ser Glu Leu Val Glu Ala Val Asp Arg 340 345 350Asp Leu Arg Lys Met Leu Ile Asn Pro Thr Ala Val Asp Pro Leu Val 355 360365 Leu Gly Val Arg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Val Gly 370375 380 His Leu Asp Leu Leu Glu Ala Ala Lys Ser Ala Leu Asp Gln Gly Gly385 390 395 400 Tyr Asn Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala Gly ValAla Leu 405 410 415 Gly Arg Cys Ile Glu Gly Ala Tyr Glu Ser Ala Ala GlnIle Tyr Asp 420 425 430 Phe Leu Thr Lys Tyr Ala Tyr Lys 435 440 93 basepairs nucleic acid single linear other nucleic acid /desc = “maizeprotox-1 intron not provided 25 GTACGCTCCT CGCTGGCGCC GCAGCGTCTTCTTCTCAGAC TCATGCGCAG CCATGGAATT 60 GAGATGCTGA ATGGATTTTA TACGCGCGCG CAG93 2606 base pairs nucleic acid single linear DNA (genomic) NO NO Betavulgaris (sugar beet) pWDC-20 (NRRL B-21650) misc_feature 2601..2606/note= ”SalI site“ misc_feature complement (1..538) /note= ”partial cDNAof sugar beet protox-1“ misc_feature 539..2606 /note= ”sugar beetprotox-1 promoter region (partial sequence of the ~ 3 kb PstI-SalIfragment subcloned from pWDC-2 26 CTGCAGGGGG AGGGAAAGAG AGACCGCGACGGTGAGGGAG GGGAGACCGC GACGGTGAGG 60 GAGGGGAGAA CGCGACGGTG AGGGAGGGGAGAACGCGATG GTGAGGGAGG GGAGAACGCG 120 ACGCGCAGGG GAGGGGGATA ACTCGACGGTGCAGGGAGGT GAGGGGGACG ACGTGACGGC 180 GCAGGGGAGG GGGGAACCGT CGCGGGAAGGGGAAGACCGG GGGGCCGACA AGGTGGTGTT 240 ACTGGGGTAG GGAGAGGCGG CGTGGAGAATAGTAACAGAG GGAGGAGTGG TGGTGCTAGG 300 GTGGAAGAAG GGTAAGAAAG AGGAAGAAAGAGAATTAACA TTATCTTAAC CAAACACCAC 360 TCTAAATCTA AGGGTTTTCT TTTCCTTTCCTCTCCTCTCC CTTTCTTGAT TCCATTCCCT 420 TTACCCCGTT GCAACCAAAC GCCCCCTTATTATGGACCGG AGGAAGTATG TAGAGATGGT 480 CACAAAACTA CTTAAGCTGG TAACTTATAAATATACTGGG TATTAAATGA ATTAAGTGGC 540 CACAAAATGA CTATAAATTA CTTCGTAATCTTTAGGAACT ATGTTGGTCA CGAAATAACA 600 TAAAACTGGT TATTTAATGG CTTTATGTAGGTACTGCATT CATAAATATA TTTCTAACAT 660 AATCGTGGTA TGTAGGTGTT TTATAACACAAGGATTAGGT TTACACCAAT GTCATTTTCA 720 TTAGAATGTA GTTAGAATCA CTTTGGAACTTTGAAGAGTG ATGACACATT TTTATTATGC 780 TTTTATGAAA TGTCTTTGTG GTTTTTATGATAGTATTGAG TTTAAGGCAA GTTGGAAGTA 840 TATGATGGAG AAGTACAGTA TATAGGTGACAATTGGTTTG CTTGTTTCTA TGAGTTGAAA 900 GATAAGTAGT ACACGACACT GAGCAATGACCTCTTCTTAG TTGTAATTTT GTCTTCTCGA 960 CGTAGTGAAA GTACAAACAA GATTATGGCTTTCAAGCTTC CAAGATAACG AGATTGTATG 1020 AATTTTGTGG TGTATTTCAC ATCATTGTTTTACGTTGGAG ACAAACTAAA ACCAATGATG 1080 AGTTTGTGGA TTCGAGATTT GCCCCTAAGTCTTATTTACC CATGGCAAGC ATGCTGAAAC 1140 ATGTTAGTCA AACTTACACA GCTACAATGTTTAGGGATTT TGAGCAAAAA ATTTGGGTAT 1200 TCTTTGGGTA CCATTATGTG AGTTGTTGACTATGGATTAA ACAAAATCAC TATATAAAGT 1260 CTGGAATGAG AAGCATCCGC AATTGACACACCATGTTACT TTGATTGTTT CAACAAGTTT 1320 ATTAGATGTA TTTGTAGGAA TTTTGAAGAGGCGGAGATGT TGTGTTATAA TTGCTTTGGG 1380 GGTGCTTCAC ATGCACTCTG TTAGTGAGACATCTTCAGCT TATATTTTAA GGCGGTTAGT 1440 GAGTATGATT TTTTTTTTTC AAACTTTTCGATTTCCATGT AATTAAAAAA GGTGTTTGAT 1500 AAATACATGT TAAGATAGCC AAGAAAAGGCAACTTTCAAA CAAATAAAAA AAATTAAGTC 1560 GCTTAATCAT TTTTCCAAGT ACTTTTTACTTTTAACACCA CTTATTACTG AATCTATAGC 1620 CGTTAAGAAT GCATTTTCAC GCTCATACATGCAAATCAAG AACCTCCTCA TTGAAGGAGA 1680 TAATTTAGTC CTCATAAACC CCGTTAAAGACATTTTTAGC ATCCAGAGAA ATTTCGATTC 1740 AGTTAAAATT GCATATATAA CCAGAGAAACAAATTCAGAT GTTAGTCAGT CCAGCTACAT 1800 AGGTCAATGC CTGAGAGTTT AAAAGAATCCGTATCCTTAA GCATAAGTAG GTATTGAGGT 1860 GAGTTACAAA GGTAAGTTAC CGGTTACGCACCACCTCCAC CAAACAAGTA TGGTTAGAAG 1920 ATACATGTAA TCGTTTATTT AGAGTACTATTTATAAAAAA CTTTTTAACT AGAAACAGTT 1980 GTTTCATTTT GATATAAGGT TAATTAGAATTCCCGAGCAA GCAAGAAGGG GATATAGAGG 2040 ATAAGGAGGG CGAGAGAGCG AGAGAGAGATGAAATCAATG GCGTTATCAA ACTGCATTCC 2100 ACAGACACAG TGCATGCCAT TGCACAGCAGCGGGCATTAC AGGGGCAATT GTATCATGTT 2160 GTCAATTCCA TGTAGTTTAA TTGGAAGACGAGGTTATTAT TCACATAAGA AGAGGAGGAT 2220 GAGCATGAGT TGCAGCACAA GCTCAGGCTCAAAGTCAGCG GTTAAAGAAG CAGGATCAGG 2280 ATCAGGATCA GGAGCAGGAG GATTGCTAGACTGCGTAATC GTTGGAGGTG GAATTAGCGG 2340 GCTTTGCATC GCGCAGGCTC TTTGTACAAAACAGTCCTCT TTATCCCCAA ATTTTATAGT 2400 GACAGAGGCC AAAGACAGAG TTGGCGGCAACATCGTCACT GTGGAGGCCG ATGGCTATAT 2460 CTGGGAGGAG GGACCCAATA GCTTCCAGCCTTCCGACGCG GTGCTCACCA TGGCGGTAAT 2520 TCTGTCTCTT CATTATTCAT AATCATAATTCAATTCAATT CAATTCCTAA CGTGGAATGT 2580 GGAATGTGGC ATGTGCGTAG GTCGAC 260631 base pairs nucleic acid single linear other nucleic acid /desc =“Pclp_P1a - plastid clpP NO NO not provided misc_feature 4..9 /note=”EcoRI restriction site“ 27 GCGGAATTCA TACTTATTTA TCATTAGAAA G 31 32base pairs nucleic acid single linear other nucleic acid /desc =”Pclp_P1b - plastid clpP NO NO not provided misc_feature 4..9 /note=“XbaI restriction site” 28 GCGTCTAGAA AGAACTAAAT ACTATATTTC AC 32 30base pairs nucleic acid single linear other nucleic acid /desc =“Pclp_P2b - plastid clpP NO NO not provided misc_feature 4..9 /note=”NcoI restriction site“ 29 GCGCCATGGT AAATGAAAGA AAGAACTAAA 30 30 basepairs nucleic acid single linear other nucleic acid /desc =”Trps16_P1a - plastid rps16 NO NO not provided misc_feature 4..9 /note=“XbaI restriction site” 30 GCGTCTAGAT CAACCGAAAT TCAATTAAGG 30 27 basepairs nucleic acid single linear other nucleic acid /desc =“Trps16_p1b - plastid rps16 NO NO not provided misc_feature 4..9 /note=”HindIII restriction site“ 31 CGCAAGCTTC AATGGAAGCA ATGATAA 27 36 basepairs nucleic acid single linear other nucleic acid /desc = ”minpsb_U -plastid psbA NO NO not provided 32 GGGAGTCCCT GATGATTAAA TAAACCAAGATTTTAC 36 40 base pairs nucleic acid single linear other nucleic acid/desc = “minpsb_L - plastid psbA NO NO not provided 33 CATGGTAAAATCTTGGTTTA TTTAATCATC AGGGACTCCC 40 32 base pairs nucleic acid singlelinear other nucleic acid /desc = ”APRTXP1a - top strand PCR NO NO notprovided misc_feature 5..10 /note= “NcoI restriction site/ATG startcodon” 34 GGGACCATGG ATTGTGTGAT TGTCGGCGGA GG 32 24 base pairs nucleicacid single linear other nucleic acid /desc = “APRTXP1b - bottom strandPCR NO NO not provided 35 CTCCGCTCTC CAGCTTAGTG ATAC 24 633 base pairsnucleic acid single linear cDNA sugar cane misc_feature 1..308 /product=”Sugar cane protox-1 partial coding sequence“ 36 TTTCCAAGAC TGAAAGTGAGCTGGTAGAAG CAGTTGACCG TGACCTCCGG AAAATGCTTA 60 TAAATCCTAC AGCAGTGGACCCTTTAGTCC TTGGTGTCCG AGTTTGGCCA CAAGCCATAC 120 CTCAGTTCCT GGTAGGACATCTTGATCTTC TGGAGGCCGC AAAATCTGCC CTGGACCGAG 180 GTGGCTACGA TGGGCTGTTCCTAGGAGGGA ACTATGTTGC AGGAGTTGCC CTAGGCAGAT 240 GCGTTGAGGG CGCGTATGAGAGTGCCTCGC AAATATATGA CTTCTTGACC AAGTATGCCT 300 ACAAGTGATG AAAGAAGTGGAGTGCTGCTT GTTAATTGTT ATGTTGCATA GATGAGGTGA 360 GACCAGGAGT AGTAAAAGCGTTACGAGTAT TTTTCATTCT TATTTTGTAA ATTGCACTTC 420 TGGTTTTTTC CTGTCAGTAATTAGTTAGAT TTTAGTTCTG TAGGAGATTG TTCTGTTCAC 480 TGCCCTACAA AAGAATTTTTATTTTGCATT CGTTTATGAG AGCTGTGCAG ACTTATGTAG 540 CGTTTTTCTG TAAGTACCAACAAAATCAAA TACTATTCTG TAAGAGCTAA CAGAATGTGC 600 AACTGAGATT GCCTTGGATGAAAAAAAAAA AAA 633 101 amino acids amino acid single linear protein notprovided 37 Ser Lys Thr Glu Ser Glu Leu Val Glu Ala Val Asp Arg Asp LeuArg 1 5 10 15 Lys Met Leu Ile Asn Pro Thr Ala Val Asp Pro Leu Val LeuGly Val 20 25 30 Arg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Val Gly HisLeu Asp 35 40 45 Leu Leu Glu Ala Ala Lys Ser Ala Leu Asp Arg Gly Gly TyrAsp Gly 50 55 60 Leu Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala Leu GlyArg Cys 65 70 75 80 Val Glu Gly Ala Tyr Glu Ser Ala Ser Gln Ile Tyr AspPhe Leu Thr 85 90 95 Lys Tyr Ala Tyr Lys 100

What is claimed is:
 1. A shuffled DNA molecule, wherein said shuffledDNA molecule encodes a protox enzyme having enhanced tolerance to aherbicide that inhibits the protox activity encoded by a template DNAmolecule from which said shuffled DNA molecule is derived.
 2. Theshuffled DNA molecule of claim 1 , wherein said herbicide is selectedfrom the group consisting of an aryluracil, a diphenylether, anoxidiazole, an imide, a phenyl pyrazole, a pyridyl pyrazole, a pyridinederivative, a 3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, aphenopylate and 0-phenylpyrrolidino- and piperidinocarbamate analogs ofsaid phenopylate.
 3. A mutagenized DNA molecule obtained by shuffling atemplate DNA molecule encoding an enzyme having protox activity, whereinsaid mutagenized DNA molecule encodes a protox enzyme having enhancedtolerance to a herbicide that inhibits the protox activity encoded bysaid template DNA molecule.
 4. The mutagenized DNA molecule of claim 3 ,wherein said herbicide is selected from the group consisting of anaryluracil, a diphenylether, an oxidiazole, an imide, a phenyl pyrazole,a pyridyl pyrazole, a pyridine derivative, a3-substituted-2-aryl4,5,6,7-tetrahydroindazole, a phenopylate and0-phenylpyrrolidino- and piperidinocarbamate analogs of saidphenopylate.
 5. A method for forming a mutagenized DNA molecule encodingan enzyme having protox activity from a template DNA molecule encodingan enzyme having protox activity, wherein said template DNA molecule hasbeen cleaved into double-stranded-random fragments, said methodcomprising the steps of: a) adding to the resultant population ofdouble-stranded-random fragments at least one single-stranded ordouble-stranded oligonucleotide, wherein said oligonucleotide comprisesan area of identity and an area or heterology to the template DNAmolecule; b) denaturing the resultant mixture of double-stranded-randomfragments and oligonucleotides into single-stranded molecules; c)incubating the resultant population of single-stranded molecules with apolymerase under conditions that result in the annealing of saidsingle-stranded molecules at said areas of identity to form pairs ofannealed fragments, said areas of identity being sufficient for onemember of a pair to prime replication of the other, thereby forming amutagenized double-stranded polynucleotide; d) repeating steps b) and c)for at least two further cycles, wherein the resultant mixture in stepb) of a further cycle includes the mutagenized double-strandedpolynucleotide from step c) of the previous cycle, and the further cycleforms a further mutagenized double-stranded polynucleotide; wherein themutagenized double-stranded polynucleotide encodes a protox enzymehaving enhanced tolerance to a herbicide that inhibits the protoxactivity encoded by the template DNA molecule.
 6. The method of claim 5, wherein said template DNA molecule is derived from a eukaryote.
 7. Themethod of claim 6 , wherein said eukaryote is a higher eukaryote.
 8. Themethod of claim 7 , wherein said higher eukaryote is a plant.
 9. Themethod of claim 8 , wherein said plant is selected from the groupconsisting of Arabidopsis thaliana, oilseed rape, soybean, sugarbeet,cotton, maize, wheat, rice, sugarcane, and sorghum.
 10. The method ofclaim 8 , wherein said template DNA molecule derived from said plantcomprises at least one mutation and encodes a modifiedprotoporphyrinogen oxidase (protox) having at least one amino acidmodification, wherein said modified protox is tolerant to a herbicide inamounts that inhibit said protox.
 11. The method of claim 10 , whereinwherein said template DNA molecule is further characterized in that atleast one of the following conditions is met: (a) said template DNAmolecule has a sequence that encodes amino acid sub-sequence APΔ₁F,wherein Δ₁ is an amino acid other than arginine; (b) said template DNAmolecule has a sequence that encodes amino acid sub-sequence FΔ₂S,wherein Δ₂ is an amino acid other than cysteine; (c) said template DNAmolecule has a sequence that encodes amino acid sub-sequence YΔ₃G,wherein Δ₃ is an amino acid other than alanine; (d) said template DNAmolecule has a sequence that encodes amino acid sub-sequence AΔ₄D,wherein Δ₄ is an amino acid other than glycine; (e) said template DNAmolecule has a sequence that encodes amino acid sub-sequence YΔ₅P,wherein Δ₅ is an amino acid other than proline; (f) said template DNAmolecule has a sequence that encodes amino acid sub-sequence PΔ₆A,wherein Δ₆ is an amino acid other than valine; (g) said template DNAmolecule has a sequence that encodes amino acid sub-sequence Δ₇IG,wherein Δ₇ is an amino acid other than tyrosine; (h) said template DNAmolecule has a sequence that encodes amino acid sub-sequence YIGGΔ₈,wherein Δ₈ is an amino acid other than alanine or serine; (i) saidtemplate DNA molecule has a sequence that encodes amino acidsub-sequence AΔ₉GP, wherein Δ₉ is an amino acid other than isoleucine;(j) said template DNA molecule has a sequence that encodes amino acidsub-sequence G₁₀A, wherein Δ₁₀ is an amino acid other than valine; (k)said template DNA molecule has a sequence that encodes amino acidsub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine, andsaid template DNA molecule also has a sequence that encodes one of thegroup consisting of: (1) sub-sequence QΔ₁₁ is, wherein Δ₁₁ is an aminoacid other than proline, (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is anamino acid other than threonine, (3) sub-sequence SWXLΔ₁₃, wherein Δ₁₃is an amino acid other than serine, (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄is an amino acid other than asparagine, and (5) sub-sequence GΔ₁₅XGL,wherein Δ₁₅ is an amino acid other than tyrosine; (l) said template DNAmolecule has a sequence that encodes amino acid sub-sequence Δ₇IG,wherein Δ₇ is an amino acid other than tyrosine, and said template DNAmolecule also has a sequence that encodes one of the group consistingof: (1) sub-sequence QΔ₁₁S, wherein Δ₁₁ is an amino acid other thanproline, (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is an amino acid otherthan threonine, (3) sub-sequence SWXLVΔ₁₃, wherein Δ₁₃ is an amino acidother than serine, (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄ is an amino acidother than asparagine, and (5) sub-sequence GΔ₁₅XGL, wherein Δ₁₅ is anamino acid other than tyrosine; and (m) said template DNA molecule has asequence that encodes amino acid sub-sequence TΔ₁₆G, wherein Δ₁₆ is anamino acid other than leucine, and said template DNA molecule also has asequence that encodes amino acid sub-sequence YVΔ₁₇G, wherein Δ₁₆ is anamino acid other than alanine.
 12. The method of claim 5 , wherein saidtemplate DNA molecule is derived from a prokaryote.
 13. The method ofclaim 5 , wherein said herbicide is selected from the group consistingof an aryluracil, a diphenylether, an oxidiazole, an imide, a phenylpyrazole, a pyridyl pyrazole, a pyridine derivative, a3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, a phenopylate and0-phenylpyrrolidino- and piperidinocarbamate analogs of saidphenopylate.
 14. A mutagenized DNA molecule encoding an enzyme havingprotox activity obtained by the method of claim 5 , wherein saidmutagenized DNA molecule encodes a protox enzyme having enhancedtolerance to a herbicide that inhibits the protox activity encoded bysaid template DNA molecule.
 15. A method for forming a mutagenized DNAmolecule encoding an enzyme having protox activity from at least twonon-identical template DNA molecules encoding enzymes having protoxactivity, said method comprising the steps of: a) adding to the templateDNA molecules at least one oligonucleotide comprising an area ofidentity to each of the template DNA molecule; b) denaturing theresultant mixture into single-stranded molecules; c) incubating theresultant population of single-stranded molecules with a polymeraseunder conditions that result in the annealing of the oligonucleotides tothe template DNA molecules, wherein the conditions for polymerization bythe polymerase are such that polymerization products corresponding to aportion of the template DNA molecules are obtained; d) repeating thesecond and third steps for at least two further cycles, wherein theextension products obtained in step c) are able to switch template DNAmolecule for polymerization in the next cycle, thereby forming amutagenized double-stranded polynucleotide comprising sequences derivedfrom different template DNA molecules; wherein the mutagenizeddouble-stranded polynucleotide encodes a protox enzyme having enhancedtolerance to a herbicide that inhibits the protox activity encoded bythe template DNA molecules.
 16. The method of claim 15 , wherein atleast one template DNA molecule is derived from a eukaryote.
 17. Themethod of claim 16 , wherein said eukaryote is a higher eukaryote. 18.The method of claim 17 , wherein said higher eukaryote is a plant. 19.The method of claim 17 , wherein said plant is selected from the groupconsisting of Arabidopsis thaliana, oilseed rape, soybean, sugarbeet,cotton, maize, wheat, rice,;sugarcane, and sorghum.
 20. The method ofclaim 17 , wherein at least one said template DNA molecule derived fromsaid plant comprises at least one mutation and encodes a modifiedprotoporphyrinogen oxidase (protox) having at least one amino acidmodification, wherein said modified protox is tolerant to a herbicide inamounts that inhibit said protox.
 21. The method of claim 20 , whereinwherein at least one said template DNA molecule is further characterizedin that at least one of the following conditions is met: (a) saidtemplate DNA molecule has a sequence that encodes amino acidsub-sequence APΔ₁F, wherein Δ₁ is an amino acid other than arginine; (b)said template DNA molecule has a sequence that encodes amino acidsub-sequence FΔ₂S, wherein Δ₂ is an amino acid other than cysteine; (c)said template DNA molecule has a sequence that encodes amino acidsub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine; (d)said template DNA molecule has a sequence that encodes amino acidsub-sequence AΔ₄D, wherein Δ₄ is an amino acid other than glycine; (e)said template DNA molecule has a sequence that encodes amino acidsub-sequence YΔ₅P, wherein Δ₅ is an amino acid other than proline; (f)said template DNA molecule has a sequence that encodes amino acidsub-sequence PΔ₆A, wherein Δ₆ is an amino acid other than valine; (g)said template DNA molecule has a sequence that encodes amino acidsub-sequence Δ₇IG, wherein Δ₇ is an amino acid other than tyrosine; (h)said template DNA molecule has a sequence that encodes amino acidsub-sequence YIGGΔ_(8,) wherein Δ₈ is an amino acid other than alanineor serine; (i) said template DNA molecule has a sequence that encodesamino acid sub-sequence AΔGP, wherein Δ₉ is an amino acid other thanisoleucine; (j) said template DNA molecule has a sequence that encodesamino acid sub-sequence AΔ₁₀A, wherein Δ₁₀ is an amino acid other thanvaline; (k) said template DNA molecule has a sequence that encodes aminoacid sub-sequence YΔ₃G, wherein Δ₃ is an amino acid other than alanine,and said template DNA molecule also has a sequence that encodes one ofthe group consisting of: (1) sub-sequence QΔ₁₁S, wherein Δ₁₁ is an aminoacid other than proline, (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is anamino acid other than threonine, (3) sub-sequence SWXLΔ₁₃, wherein Δ₁₃is an amino acid other than serine, (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄is an amino acid other than asparagine, and (5) sub-sequence GΔ₁₅XGL,wherein Δ₁₅ is an amino acid other than tyrosine; (l) said template DNAmolecule has a sequence that encodes amino acid sub-sequence Δ₇IG,wherein Δ₇ is an amino acid other than tyrosine, and said template DNAmolecule also has a sequence that encodes one of the group consistingof: (1) sub-sequence QΔ₁₁S, wherein Δ₁₁ is an amino acid other thanproline, (2) sub-sequence IGGΔ₁₂, wherein Δ₁₂ is an amino acid otherthan threonine, (3) sub-sequence SWXLΔ₁₃, wherein Δ₁₃ is an amino acidother than serine, (4) sub-sequence LΔ₁₄Y, wherein Δ₁₄ is an amino acidother than asparagine, and (5) sub-sequence GΔ₁₅XGL, wherein Δ₁₅ is anamino acid other than tyrosine; and (m) said template DNA molecule has asequence that encodes amino acid sub-sequence TΔ₁₆G, wherein Δ₁₆ is anamino acid other than leucine, and said template DNA molecule also has asequence that encodes amino acid sub-sequence YVΔ₁₇G, wherein Δ₁₆ is anamino acid other than alanine.
 22. The method of claim 15 , wherein atleast one said template DNA molecule is derived from a prokaryote. 23.The method of claim 15 , wherein said herbicide is selected from thegroup consisting of an aryluracil, a diphenylether, an oxidiazole, animide, a phenyl pyrazole, a pyridyl pyrazole, a pyridine derivative, a3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, a phenopylate and0-phenylpyrrolidino- and piperidinocarbamate analogs of saidphenopylate.
 24. A mutagenized DNA molecule encoding an enzyme havingprotox activity obtained by the method of claim 15 , wherein saidmutagenized DNA molecule encodes a protox enzyme having enhancedtolerance to a herbicide that inhibits the protox activity encoded bysaid template DNA molecule.
 25. The method of claim 15 , wherein saidherbicide is selected from the group consisting of an aryluracil, adiphenylether, an oxidiazole, an imide, a phenyl pyrazole, a pyridylpyrazole, a pyridine derivative, a3-substituted-2-aryl-4,5,6,7-tetrahydroindazole, a phenopylate and0-phenylpyrrolidino- and piperidinocarbamate analogs of saidphenopylate.
 26. An isolated DNA fragment capable of specificallyhybridizing to a eukaryotic protoporphyrinogen oxidase gene or mRNA,wherein said DNA fragment comprises a contiguous portion of a codingsequence for a protoporphyrinogen oxidase from a eukaryote at least 10nucleotides in length.
 27. The isolated DNA fragment of claim 26 ,wherein said coding sequence is selected from the group consisting ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 23, and 36.